Devices and methods for using centripetal acceleration to drive fluid movement in a microfluidics system for performing biological fluid assays

ABSTRACT

This invention provides methods and apparatus for performing microanalytic and microsynthetic analyses and procedures. Specifically, the invention provides a microsystem platform for use with a micromanipulation device to manipulate the platform by rotation, thereby utilizing the centripetal force resulting from rotation of the platform to motivate fluid movement through microchannels embedded in the microplatform. The microsystem platforms of the invention are also provided having microfluidics components, resistive heating elements, temperature sensing elements, mixing structures, capillary and sacrificial valves, and methods for using these microsystems platforms for performing biological, enzymatic, immunological and chemical assays.

[0001] This application claim priority to U.S. Ser. No. 09/083,678,filed May 22, 1998. This application is also related to U.S. Ser. No.08/995,056, filed Dec. 19, 1997, U.S. Ser. No. 08/910,726, filed Aug.12, 1997, U.S. Ser. No. 08/768,990, filed Dec. 18, 1996 and U.S. Ser.No. 08/761,063, filed Dec. 5, 1996, the disclosures of every one ofwhich are explicitly incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to methods and apparatus for performingmicroanalytic analyses and procedures on fluid samples. In particular,the invention relates to microminiaturization of genetic, biochemicaland chemical processes related to analysis, synthesis and purification.Specifically, the invention provides a microsystem platform that isrotationally manipulated by a micromanipulation device, therebyutilizing the centripetal forces resulting from rotation of the platformto motivate fluid movement through microchannels embedded in themicroplatform. The microsystem platforms of the invention are providedhaving microfluidics components, chambers and reservoirs, resistiveheating elements, temperature sensing elements, mixing structures,capillary and sacrificial valves, and methods for using thesemicrosystems platforms for performing biological, enzymatic,immunological and chemical assays.

[0004] 2. Summary of the Related Art

[0005] Assays for detecting analytes in fluid samples, particularlycomplex fluids such as biological fluid samples, are used for a varietyof diagnostic, environmental, synthetic and analytical purposes in themedical, biological, chemical, biochemical and environmental arts.

[0006] Certain analytes are important for diagnosis and monitoring ofacute and chronic disease in humans. For example, diabetes is the fourthmajor cause of morbidity and mortality in the U.S., even though thebiochemical basis for the disease has been known for at least a centuryand drugs (primarily insulin) and methods for managing the disease arerobust and widely used. It has been recognized that sensitive monitoringof blood sugar levels, either directly or by detecting levels ofmetabolites and disease-associated modifications (such as the relativefraction of glycated hemoglobin in the bloodstream) is important inmanaging the disease. However, fast and inexpensive ways of performingthe assays necessary for efficient management of multiple indicators ofthe disease state are not currently available.

[0007] In the field of medical, biological and chemical assays,mechanical and automated fluid handling systems and instruments areknown in the prior art.

[0008] U.S. Pat. No. 4,279,862, issued Jul. 21, 1981 to Bertaudiere etal. disclose a centrifugal photometric analyzer.

[0009] U.S. Pat. No. 4,381,291, issued Apr. 26, 1983 to Ekins teachanalytic measurement of free ligands.

[0010] U.S. Pat. No. 4,515,889, issued May 7, 1985 to Klose et al. teachautomated mixing and incubating reagents to perform analyticaldeterminations.

[0011] U.S. Pat. No. 4,676,952, issued Jun. 30, 1987 to Edelmann et al.teach a photometric analysis apparatus.

[0012] U.S. Pat. No. 4,745,072, issued May 17, 1998 to Ekins disclosesimmunoassay in biological fluids.

[0013] U.S. Pat. No. 5,061,381, issued Oct. 29, 1991 to Burd discloses acentrifugal rotor for performing blood analyses.

[0014] U.S. Pat. No. 5,122,284, issued Jun. 16, 1992 to Braynin et al.discloses a centrifugal rotor comprising a plurality of peripheralcuvettes.

[0015] U.S. Pat. No. 5,160,702, issued Nov. 3, 1993 to Kopf-Sill and Zukdiscloses rotational frequency-dependent

valves

using capillary forces and siphons, dependent on

wettablility” of liquids used to prime said siphon.

[0016] U.S. Pat. No. 5,171,695, issued Dec. 15, 1992 to Ekins disclosesdetermination of analyte concentration using two labeling markers.

[0017] U.S. Pat. No. 5,173,193, issued Dec. 22, 1992 to Schembridiscloses a centrifugal rotor for delivering a metered amount of a fluidto a receiving chamber on the rotor.

[0018] U.S. Pat. No. 5,242,803, issued Sep. 7,1993 to Burtis et al.disclose a rotor assembly for carrying out an assay.

[0019] U.S. Pat. No. 5,409,665, issued Apr. 25, 1995 to Burd discloses acuvette filling in a centrifuge rotor.

[0020] U.S. Pat. No. 5,413,009, issued Jul. 11, 1995 to Ekins disclosesa method for analyzing analytes in a liquid.

[0021] U.S. Pat. No. 5,472,603, issued Dec. 5, 1995 to Schembridiscloses an analytical rotor comprising a capillary passage having anexit duct wherein capillary forces prevent fluid flow at a givenrotational speed and permit flow at a higher rotational speed.

[0022] Anderson, 1968, Anal. Biochem. 28: 545-562 teach a multiplecuvette rotor for cell fractionation.

[0023] Renoe et al., 1974 Clin. Chem. 20: 955-960 teach a

minidisc

module for a centrifugal analyzer.

[0024] Burtis et al., 1975, Clin. Chem. 20: 932-941 teach a method for adynamic introduction of liquids into a centrifugal analyzer.

[0025] Fritsche et al., 1975, Clin. Biochem. 8: 240-246 teach enzymaticanalysis of blood sugar levels using a centrifugal analyzer.

[0026] Burtis et al., 1975, Clin Chem. 21: 1225-1233 teach amultipurpose optical system for use with a centrifugal analyzer.

[0027] Hadjiioannou et al., 1976, Clin. Chem. 22: 802-805 teachautomated enzymatic ethanol determination in biological fluids using aminiature centrifugal analyzer.

[0028] Lee et al., 1978, Clin. Chem. 24: 1361-1365 teach a automatedblood fractionation system.

[0029] Cho et al., 1982, Clin. Chem. 28: 1956-1961 teach a multichannelelectrochemical centrifugal analyzer.

[0030] Bertrand et al., 1982, Clinica Chimica Acta 119: 275-284 teachautomated determination of serum 5′-nucleotidase using a centrifugalanalyzer.

[0031] Schembri et al., 1992, Clin Chem. 38: 1665-1670 teach a portablewhole blood analyzer.

[0032] Walters et al., 1995, Basic Medical Laboratory Technologies, 3rded., Delmar Publishers: Boston teach a variety of automated medicallaboratory analytic techniques.

[0033] Recently, microanalytical devices for performing select reactionpathways have been developed.

[0034] U.S. Pat. No. 5,006,749, issued Apr. 9, 1991 to White disclosemethods apparatus for using ultrasonic energy to move microminiatureelements.

[0035] U.S. Pat. No. 5,252,294, issued Oct. 12, 1993 to Kroy et al.teach a micromechanical structure for performing certain chemicalmicroanalyses.

[0036] U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Wilding et al.teach fluid handling on microscale analytical devices.

[0037] U.S. Pat. No. 5,368,704, issued Nov. 29, 1994 to Madou et al.teach microelectrochemical valves.

[0038] International Application, Publication No. WO93/22053, published11 Nov. 1993 to University of Pennsylvania disclose microfabricateddetection structures.

[0039] International Application, Publication No. WO93/22058, published11 Nov. 1993 to University of Pennsylvania disclose microfabricatedstructures for performing polynucleotide amplification.

[0040] Columbus et al., 1987, Clin. Chem. 33: 1531-1537 teach fluidmanagement of biological fluids.

[0041] Ekins et al., 1994 Ann. Biol. Clin. 50: 337-353 teach amultianalytic microspot immunoassay.

[0042] Wilding et al., 1994, Clin. Chem. 40: 43-47 disclose manipulationof fluids on straight channels micromachined into silicon.

[0043] One drawback in the prior art microanalytical methods andapparati has been the difficulty in designing systems for moving fluidson microchips through channels and reservoirs having diameters in the10-500 μm range. Microfluidic systems require precise and accuratecontrol of fluid flow and valving to control chemical reactions andanalyte detection. Conventional pumping and valving mechanisms have beendifficult to incorporate into microscale structures due to inherentconflicts-of-scale. These conflicts of scale arise in part due to thefact that molecular interactions arising out of mechanical components ofsuch components, which are negligible in large (macroscopic) scaledevices, become very significant for devices built on a microscopicscale.

[0044] While devices and pumping and valving mechanisms have beendeveloped which overcome some of these conflict-of-scale difficulties,there are other inherent problems with these systems. A number ofmicroanalytical platforms have been developed which use electrokineticforces for fluid pumping: electroosmotic flow devices;electrohydrodynamic devices; and electrophoretic devices. An inherentdrawback in these systems is that they rely on precise control of pH andfree charges in the fluid being pumped. This makes them incapable ofpumping most raw biological samples, such as blood and urine, andcreates difficulties in pumping organic solvents. In cases where thesesystems may be used, pre-conditioning of the fluid to enhanceelectrokinetic effects is usually required.

[0045] Systems that use centripetal force to effect fluid movement inmicrostructures address the need for a pumping mechanism to effect fluidflow, but cannot alone solve these scale-related drawbacks ofconventional fluidics reduced to microfluidics scale. There remains aneed for a simple, flexible, reliable, rapid and economicalmicroanalytic and microsynthetic reaction platform for performingbiological, biochemical and chemical analyses and syntheses that canmove fluids within the structural components of a microsystems platform.Such a platform should be able to move nanoliter-to microliter amountsof fluid, including reagents and reactants, at rapid rates to effect theproper mixing of reaction components, removal of reaction side products,and isolation of desired reaction products and intermediates. Thereremains a need in the art for centripetally-motivated microfluidicsplatforms capable of precise and accurate control of flow and meteringof fluids in both microchip-based and centrifugal microplatform-basedtechnologies.

SUMMARY OF THE INVENTION

[0046] This invention provides microsystems platforms as disclosed inco-owned and co-pending U.S. Ser. No. 08/761,063, filed Dec. 5, 1996 andU.S. Ser. No. 09/083,678, filed May 22, 1998, each of which isincorporated by reference herein. Specifically, this invention providesmicrofluidics platforms for performing biological, enzymatic,immunological and chemical assays.

[0047] It is an advantage of the centrifugal rotors and Microsystemsplatforms of the invention that an imprecise amount of a fluidcomprising a biological sample can be applied to the rotor or platformand a precise volumetric amount of the biological sample is delivered toa fluid reservoir comprising a reaction vessel or other component of therotor or platform for performing chemical, biochemical, immunological orother analyses. It is an advantage of the centrifugal rotors andMicrosystems platforms of the invention that metering of said preciseamount of a biological fluid sample, for example, a drop of blood, isprovided as an intrinsic property of the metering capillary channel ofthe rotor or platform, thereby avoiding variability introduced bycentripetal metering of the sample into a reaction reservoir. It is afurther advantage of the centrifugal rotors and Microsystems platformsof the invention that an operator can avoid having to precisely measurean amount of a fluid comprising a biological sample for application tothe rotor or microsystem platform, thereby permitting end-users,including consumers, having a lower level of sophistication to use amedically diagnostic or other embodiment of the rotor or microsystemplatform of the invention.

[0048] It is an advantage of the centrifugal rotors and Microsystemsplatforms of the invention that fluid movement into and out of fluidreservoirs on the rotor or platform is precisely determined bydisplacement of a first fluid, such as biological sample, from a fluidreservoir by a second fluid contained in a second reservoir on the rotoror platform. It is also an advantage of the centrifugal rotors andMicrosystems platforms of the invention that approximately completereplacement of the volumetric capacity of a first reservoir can beachieved by using fluid displacement as disclosed herein, therebyproviding for maximum recovery of a first fluid sample upon displacementby a second fluid, or maximum delivery and replacement of the firstfluid by the second fluid. This aspect of the invention is advantageousfor providing sequential chemical or biochemical reaction steps whereinmixing of the reagents is not desired.

[0049] It is also an advantage of the centrifugal rotors andMicrosystems platforms of the invention that these platforms provide anintegrated microfluidics system containing components and structures forperforming microanalytic assays whereby fluid flow on the platform ismotivated by centripetal force and controlled by capillary and/orsacrificial valves. The invention provides such integrated platformswhereby an operator is required simply to apply a sample, mostpreferably an imprecise volume of a fluid sample, to an entry port onthe disk surface, and a complex series of analytical steps are performedwithout further operator manipulation on the platform. Movement offluids on the disk, and the sequence of analytical reaction stepsperformed thereupon, is a consequence of changes in rotor speed and/orthe opening of sacrificial valves as directed by an instruction setcontained in a program contained on the disk itself or in the memory ofthe micromanipulation apparatus that controls disk rotation andperformance.

[0050] The Microsystems platforms also provide disks that can perform amultiplicity of analytical reactions on either several samples or aparticular sample, whereby the reactions are performed sequentially orindividually. In addition, a wide variety of analytic reactions can beperformed on the Microsystems platforms of the invention, as furtherdescribed below.

[0051] Specific preferred embodiments of the present invention willbecome evident from the following more detailed description of certainpreferred embodiments and the claims.

DESCRIPTION OF THE DRAWINGS

[0052]FIGS. 1 through 8 are schematic representations of microfluidicsarrays and components for performing direct analyte detection assaysusing the Microsystems platforms of the invention.

[0053]FIGS. 9 through 11 are schematic representations of microfluidicsarrays and components for performing separations of analyte from a fluidsample using the Microsystems platforms of the invention.

[0054]FIGS. 12 and 13 are schematic representations of microfluidicsarrays and components for performing both direct analyte detectionassays and analyte separations using the Microsystems platforms of theinvention.

[0055]FIGS. 14A and 14B illustrate the results of the assays disclosedin Example 4 FIG. 15 is a photograph showing DNA recovery using theassays described in Example 4.

[0056]FIG. 16 is a schematic diagram of a snap-in glucose assaycomponent of a Microsystems platform of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0057] The present invention provides centrifugal rotors andMicrosystems platforms for providing centripetally-motivated fluidmicromanipulation.

[0058] For the purposes of this invention, the term “sample” will beunderstood to encompass any fluid, solution or mixture, either isolatedor detected as a constituent of a more complex mixture, or synthesizedfrom precursor species.

[0059] For the purposes of this invention, the term “in fluidcommunication” or “fluidly connected” is intended to define componentsthat are operably interconnected to allow fluid flow between components.In preferred embodiments, the platform comprises a rotatable platform,more preferably a disk, whereby fluid movement on the disk is motivatedby centripetal force upon rotation of the disk.

[0060] For the purposes of this invention, the term “a centripetallymotivated fluid micromanipulation apparatus” is intended to includeanalytical centrifuges and rotors, microscale centrifugal separationapparati, and most particularly the microsystems platforms and diskhandling apparati of International Application No. WO97/21090,incorporated by reference.

[0061] For the purposes of this invention, the term “Microsystemsplatform” is intended to include centripetally-motivated microfluidicsarrays as disclosed in International Application No. WO97/21090.

[0062] For the purposes of this invention, the terms “capillary”,“microcapillary and “microchannel” will be understood to beinterchangeable and to be constructed of either wetting or non-wettingmaterials where appropriate.

[0063] For the purposes of this invention, the term “fluid chamber” willbe understood to mean a defined volume on a rotor or Microsystemsplatform of the invention comprising a fluid.

[0064] For the purposes of this invention, the term “entry port” will beunderstood to mean a undefined volume on a rotor or microsystemsplatform of the invention comprising a means for applying a fluid to therotor or platform.

[0065] For the purposes of this invention, the term “capillary junction”will be understood to mean a junction of two components wherein one orboth of the lateral dimensions of the junction are larger than thecorresponding dimensions of the capillary. In wetting or wettablesystems, such junctions are where capillary valving occurs, becausefluid flow through the capillaries is stopped at such junctions. Innon-wetting or non-wettable junctions, the exit from the chamber orreservoir is where the capillary junction occurs. In general, it will beunderstood that capillary junctions are formed when the dimensions ofthe components change from a small diameter (such as a capillary) to alarger diameter (such as a chamber) in wetting systems, in contrast tonon-wettable systems, where capillary junctions form when the dimensionsof the components change from a larger diameter (such as a chamber) to asmall diameter (such as a capillary).

[0066] For the purposes of this invention, the term “biological sample”or “biological fluid sample” will be understood to mean anybiologically-derived analytical sample, including but not limited toblood, plasma, serum, lymph, saliva, tears, cerebrospinal fluid, urine,sweat, plant and vegetable extracts, semen, cell culture fluids,cellular lysate aqueous or non-aqueous fractions of the above andascites fluid.

[0067] For the purposes of this invention, the term “air displacementchannels” will be understood to include ports in the surface of theplatform that are contiguous with the components (such as chambers andreservoirs) on the platform, and that comprise vents and microchannelsthat permit displacement of air from components of the platforms androtors by fluid movement.

[0068] For the purposes of this invention, the term “capillary action”will be understood to mean fluid flow in the absence of rotationalmotion or centripetal force applied to a fluid on a rotor or platform ofthe invention.

[0069] For the purposes of this invention, the term “capillarymicrovalve” will be understood to mean a capillary comprising acapillary junction whereby fluid flow is impeded and can be motivated bythe application of pressure on a fluid, typically by centripetal forcecreated by rotation of the rotor or platform of the invention.

[0070] The microplatforms of the invention (preferably and hereinaftercollectively referred to as discs or disks for the purposes of thisinvention), the terms “microplatform”, “Microsystems platform” and“disc” or “disk” are considered to be interchangeable, and are providedto comprise one or a multiplicity of microsynthetic or microanalyticsystems. Such microsynthetic or microanalytic systems in turn comprisecombinations of related components as described in further detail hereinthat are operably interconnected to allow fluid flow between componentsupon rotation of the disk. These components can be fabricated asdescribed below either integral to the disk or as modules attached to,placed upon, in contact with or embedded in the disk. The invention alsocomprises a micromanipulation device for manipulating the disks of theinvention, wherein the disk is rotated within the device to providecentripetal force to effect fluid flow on the disk. Accordingly, thedevice provides means for rotating the disk at a controlled rotationalvelocity, for stopping and starting disk rotation, and advantageouslyfor changing the direction of rotation of the disk. Bothelectromechanical means and control means, as further described herein,are provided as components of the devices of the invention. Userinterface means (such as a keypad and a display) are also provided, asfurther described in International Application WO97/21090.

[0071] Fluid (including reagents, samples and other liquid components)movement is controlled by centripetal acceleration due to rotation ofthe platform. The magnitude of centripetal acceleration required forfluid to flow at a rate and under a pressure appropriate for aparticular microsystem is determined by factors including but notlimited to the effective radius of the platform, the position angle ofthe structures on the platform with respect to the direction of rotationand the speed of rotation of the platform.

[0072] The capillary junctions and microvalves of the invention arebased on the use of rotationally-induced fluid pressure to overcomecapillary forces. Fluids which completely or partially wet the materialof the microchannels (or reservoirs, reaction chambers, detectionchambers, etc.) which contain them experience a resistance to flow whenmoving from a microchannel of narrow cross-section to one of largercross-section, while those fluids which do not wet these materialsresist flowing from microchannels (or reservoirs, reaction chambers,detection chambers, etc.) of large cross-section to those with smallercross-section. This capillary pressure varies inversely with the sizesof the two microchannels (or reservoirs, reaction chambers, detectionchambers, etc., or combinations thereof), the surface tension of thefluid, and the contact angle of the fluid on the material of themicrochannels (or reservoirs, reaction chambers, detection chambers,etc.) Generally, the details of the cross-sectional shape are notimportant, but the dependence on cross-sectional dimension results inmicrochannels of dimension less than 500 μm exhibit significantcapillary pressure. By varying the intersection shapes, materials andcross-sectional areas of the components of the Microsystems platform ofthe invention, valves are fashioned that require the application of aparticular pressure on the fluid to induce fluid flow. This pressure isapplied in the disks of the invention by rotation of the disk (which hasbeen shown above to vary with the square of the rotational frequency,with the radial position and with the extent of the fluid in the radialdirection). By varying capillary valve cross-sectional dimensions aswell as the position and extent along the radial direction of the fluidhandling components of the microsystem platforms of the invention,capillary valves are formed to release fluid flow in arotation-dependent manner, using rotation rates of from 100 rpm toseveral thousand rpm. This arrangement allows complex, multistep fluidprocesses to be carried out using a pre-determined, monotonic increasein rotational rate. The theoretical principles underlying the use ofcapillary junctions and microvalves are disclosed in InternationalPatent Application, Publication No. WO98/07019, incorporated byreference.

[0073] The instant invention provides Microsystems platforms comprisingmicrofluidics components, heating elements, temperature sensingelements, capillary valves, sacrificial valves and a rotor design fortransmitting electrical signals to and from the Microsystems platformsof the invention. The invention provides fluidics components forcapillary metering of precise amounts of a volume of a fluid sample fromthe application of a less precise volume of a fluid sample at an entryport on the microsystem platform. These embodiments of the inventionprovide for delivery of precise amounts of a sample such as a biologicalfluid sample without requiring a high degree of precision or accuracy bythe operator or end-user in applying the fluid to the platform, and isadvantageous in embodiments of the Microsystems platforms of theinvention that are used by consumers and other relativelyunsophisticated users. The invention also provides laminarflow-dependent replacement of a fluid in a first chamber by a seconddisplacement fluid in a second chamber on the platform. Theseembodiments of the invention provide approximately complete replacementof a fluid in one chamber on the platform with fluid from another, andthereby provide means for practicing sequential chemical reactions andother sequential processes on the platform under conditions whereinmixing of the two fluids is disadvantageous. The invention also providesturbulent flow mixing components, which permit thorough mixing ofdifferent fluid components on the platform, and in particular, theinvention provides mixing chambers fluidly connected with fluidreservoirs containing equal amounts of two or more different fluids orunequal amounts of two or more different fluids. In addition, theinvention provides fluid reservoirs fluidly connected with mixingchamber of the invention and shaped to determine the relative rate offlow of each of the different fluids into the mixing chamber. In theseembodiments, gradients of two fluids differing in viscosity, soluteconcentration or concentration of suspended particulates can be producedusing the mixing chambers of the invention, as disclosed; U.S. Ser. No.09/083,678 incorporated by reference. Such gradients can be transferredto reservoirs on the platform for further analytical manipulations, andcan form the basis for controlled testing of concentration-dependenteffects of various catalysts, drugs, toxins or other biological orchemical agents.

[0074] Platforms of the invention such as disks and the componentscomprising such platforms are advantageously provided having a varietyof composition and surface coatings appropriate for a particularapplication. Platform composition will be a function of structuralrequirements, manufacturing processes, and reagentcompatibility/chemical resistance properties. Specifically, platformsare provided that are made from inorganic crystalline or amorphousmaterials, e.g. Silicon, silica, quartz, inert metals, or from organicmaterials such as plastics, for example, poly(methyl methacrylate)(PMMA), acetonitrile-butadiene-styrene (ABS), polycarbonate,polyethylene, polystyrene, polyolefins, polypropylene and metallocene.These may be used with unmodified or modified surfaces. Surfaceproperties of these materials may be modified for specific applications.Surface modification can be achieved by silanization, ion implantationand chemical treatment with inert-gas plasmas (i.e., gases through whichelectrical currents are passed to create ionization). Also provided bythe invention are platforms made of composites or combinations of thesematerials, for example, platforms manufactures of a plastic materialhaving embedded therein an optically transparent glass surfacecomprising for example the detection chamber of the platform.Microplatform disks of the invention are preferably fabricated fromthermoplastics such as teflon, polyethylene, polypropylene,methylmethacrylates and polycarbonates, among others, due to their easeof molding, stamping and milling. Alternatively, the disks can be madeof silica, glass, quartz or inert metal. A fluid handling system isbuilt by sequential application of one or more of these materials laiddown in stepwise fashion onto the thermoplastic substrate.Alternatively, the entire disc can be injection molded, embossed orstamped. Disks of the invention are fabricated with an injection molded,optically-clear base layer having optical pits in the manner of aconventional compact disk (CD). The optical pits provide means forencoding instrument control programming, user interface information,graphics, data analysis, and sound specific to the application anddriver configuration. The driver configuration depends on whether themicromanipulation device is a handheld, benchtop or floor model, andalso on the details of external communication and other specifics of thehardware configuration. This layer is then overlaid with a reflectivesurface, with appropriate windows for external detectors, specificallyoptical detectors, being left clear on the disk. Other layers ofpolycarbonate of varying thickness are laid down on the disk in the formof channels, reservoirs, reaction chambers and other structures,including provisions on the disk for valves and other control elements.These layers can be pre-fabricated and cut with the appropriategeometries for a given application and assembled on the disk. Layerscomprising materials other than polycarbonate can also be incorporatedinto the disk. The composition of the layers on the disk depend in largepart on the specific application and the requirements of chemicalcompatibility with the reagents to be used with the disk. Electricallayers can be incorporated in disks requiring electric circuits, such aselectrophoresis applications and electrically-controlled valves. Controldevices, such as integrated circuits, laser diodes, photodiodes andresistive networks that can form selective heating areas or flexiblelogic structures can be incorporated into appropriately wired recesses,either by direct fabrication of modular installation onto the disk.Reagents that can be stored dry can be introduced into appropriate openchambers by spraying into reservoirs using means similar to inkjetprinting heads, and then dried on the disk. A top layer comprisingaccess ports and air vents, ports or shafts is then applied. Liquidreagents are then injected into the appropriate reservoirs, followed byapplication of a protective cover layer comprising a thin plastic film.

[0075] The platforms of the invention are preferably provided with amultiplicity of components, either fabricated directly onto theplatform, or placed on the platform as prefabricated modules. Inaddition to the integral components, certain devices and elements can belocated external to the platform, optimally positioned on a device ofthe invention in relation to the platform, or placed in contact with theplatform either while rotating or when at rest. Components optimallycomprising the platforms of the invention or a controlling device incombination therewith include detection chambers, reservoirs, valvingmechanisms, detectors, sensors, temperature control elements, filters,mixing elements, and control systems.

[0076] This invention provides microsystems platforms comprising thefollowing components.

1. Fluidics Components

[0077] The platforms of the invention are provided comprisingmicrofluidics handling structures in fluidic contract with one another.In preferred embodiments, fluidic contact is provided by capillary ormicrochannels comprising the surface of the platforms of the invention.Microchannel sizes are optimally determined by specific applications andby the amount of delivery rates required for each particular embodimentof the platforms and methods of the invention. Microchannel sizes canrange from 0.02 mm to a value close to the thickness of the platform.Microchannel shapes can be trapezoid, circular or other geometric shapesas required. Microchannels preferably are embedded in a platform havinga thickness of about 0.1 to 100 mm, wherein the cross-sectionaldimension of the microchannels across the thickness dimension of theplatform is less than 500 μm-800 μm and from 1 to 90 percent of saidcross-sectional dimension of the platform. In these embodiments, whichis based on the use of rotationally-induced fluid pressure to overcomecapillary forces, it is recognized that fluid flow is dependent on theorientation of the surfaces of the components. Fluids which completelyor partially wet the material of the microchannels, reservoirs,detection chambers, etc. (i.e., the components) of the platforms of theinvention which contain them experience a resistance to flow when movingfrom a component of narrow cross-section to one of larger cross-section,while those fluids which do not wet these materials resist flowing fromcomponents of the platforms of the invention of large cross-section tothose with smaller cross-section. This capillary pressure variesinversely with the sizes of the two components, or combinations thereof,the surface tension of the fluid, and the contact angle of the fluid onthe material of the components. Generally, the details of thecross-sectional shape are not important, but the dependence oncross-sectional dimension results in microchannels of dimension lessthan 500 μm exhibit significant capillary pressure. By varying theintersection shapes, materials and cross-sectional areas of thecomponents of the platform of the invention, “valves” are fashioned thatrequire the application of a particular pressure on the fluid to inducefluid flow. This pressure is applied in the disks of the invention byrotation of the disk (which has been shown above to vary with the squareof the rotational frequency, with the radial position and with theextent of the fluid in the radial direction). By varying capillary valvecross-sectional dimensions as well as the position and extent along theradial direction of the fluid handling components of the platforms ofthe invention, capillary valves are formed to release fluid flow in arotation-dependent manner, using rotation rates of from 100 rpm toseveral thousand rpm. This arrangement allows complex, multistep fluidprocesses to be carried out using a predetermined, monotonic increase inrotational rate.

[0078] A first example of the microfluidics arrays provided by thisinvention is shown in FIGS. 1A through 1D. A Microsystems platform isprovided by the invention that is specifically designed for performingan assay for detecting a chemical species in a complex mixture,preferably an aqueous mixture. These Figures illustrate an arrayadvantageously used for any such assay; detection of blood glucoseconcentration is illustrated herein. It will be understood that in thisdescription, the use of the words “cell” and “particulate” will beinterchangeable, and that cells are a particular example of a specificparticulate species comprising the fluid samples, and most preferablythe biological fluid samples, analyzed using the present invention.

[0079] A Microsystems platform provided by the invention andspecifically designed for performing blood glucose assay is illustratedin FIG. 1A. Disk embodiments of the platforms of the invention werefashioned, for example, from machined acrylic or injection-moldedpolycarbonate, polystyrene, polypropylene,acetonitrile-butadiene-styrene, or high-density polyethylene (HDPE). Theoverall disc dimensions include an outer radius of from about 1 cm toabout 15 cm and an inner radius of from about 0.1 cm to about 1 cm,wherein the disk was mounted on the spindle of a rotary device. Thethickness of the disc ranged from about 0.2 mm to about 1.5 cm. Allsurfaces coming into contact with blood on the platform may beadvantageously treated with heparin, EDTA or other anticoagulants tofacilitate fluid flow thereupon

[0080] The components of the blood glucose assay were prepared asfollows. Fluid sample entry port 101 having a depth in the platformsurface from about 0.15 cm to about 3 mm and lateral dimensions of fromabout 0.1 cm to about 2.5 cm were constructed on the platform, anddesigned to accommodate a volume of from about 5 to about 200 μL. Thisentry port was fluidly connected with a metering capillary 102 having asquare cross-sectional diameter of from about 0.02 mm to about 2 cm andproximal ends rounded with respect to entry port 101; the length of thismetering chamber array was sufficient to contain a total volume of fromabout 15 to about 150 μL. The entry port was also constructed to befluidly connected with an overflow capillary 121 having across-sectional diameter of from about 0.02 mm to about 2 mm andproximal ends rounded with respect to entry port 101. The overflowcapillary was fluidly connected with an overflow chamber 122 having adepth in the platform surface of from about 0.02 mm to about 9 mm,greater than the depth of the overflow capillary 121. Each of thechambers on the platform were also connected with air ports or airchannels, such as 114, that have dimensions of from about 0.02 mm toabout 2 mm deep and permitted venting of air displaced by fluid movementon the platform. A capillary junction 115 that is from about 0.03 mm toabout 2.2 mm deep is present in the air channel to prevent fluid flowinto the air channel.

[0081] Entry port 101 was positioned on the platform from about 1 cm toabout 12 cm from the center of rotation. Metering chamber 102 extendedfrom about 0.25 cm to about 2 cm from entry port 101. The extent of thelength of overflow capillary 121 was greater than the extent of thelength of metering capillary 102. The position of overflow chamber 122was, for example, from about 1.2 cm to about 14 cm from the axis ofrotation.

[0082] In an alternative embodiment of the fluid metering structures ofthe invention, shown in FIG. 1B, fluid sample entry port 101 iscomprised of a funnel having a depth in the platform surface from about0.75 mm to 5 mm, lateral dimensions of from about 2 mm to 2 cm in thelong dimension and from about 2 mm to 2 cm in the short dimension andpositioned from about 0.75 cm to about 5 cm from the center of rotation,and designed to accommodate a volume of about 5 to about 200 μL. Thebottom surface of the entry port consists of a slot 120 fluidlyconnected to microfluidics structures formed in the other (under) sideof the microfluidics surface of the platform. Entry port 101 isconnected to entry passageway 102 having a rectangular cross-sectionaldiameter of from about 0.1 mm to 5 mm wide and from about 0.1 mm to 5 mmdeep and extending from about 0.1 cm to about 3 cm from entry port 101.The entry passageway 102 is fluidly connected to blood entry chamber103. Blood entry chamber 103 has a depth of from about 0.1 mm to 5 mm,lateral dimensions of from about 1 mm to about 4 cm, and is positionedfrom about 0.75 cm to about 2.5 cm from the center of rotation. Bloodentry chamber 103 further comprises blood metering volume 104 designedto accommodate a volume of from about 1 μL to about 50 μL and anoverflow passageway 105. The overflow passageway was fluidly connectedwith an overflow capillary 106 having a depth in the platform surface offrom about 0.1 mm to about 1 mm. Overflow capillary 121 is furtherfluidly connected with overflow chamber 122 which is comprised of twoparts, a shallow outer portion 108 having a depth from about 0.05 mm toabout 0.5 mm and a deeper inner portion 109 having a depth of from about0.1 mm to 5 mm. Overflow chamber 122 is positioned from about 4 cm toabout 5.8 cm from the axis of rotation. The distal end of the bloodoverflow capillary 121 is chosen to be farther from the center ofrotation than the distal end of blood chamber 104. Each of the chamberson the platform were also connected with air ports or air channels 114,that are from about 0.1 mm to about 5 mm deep and permit venting of airdisplaced by fluid movement on the platform. A capillary junction 115that is from about 0.03 mm to about 2.2 mm deep is present in the airchannel to prevent fluid flow into the air channel. In alternativeembodiments, these vents may be multiply connected to one anotherthrough a manifold such that fluid flow merely displaces air within thestructure, rather than forcing it through vents in the platform surface.

[0083] Alternatively, an unmetered amount of a blood sample is placeddirectly in blood fluid chamber 104, which in this embodiment is open tothe surface of the disk to accept blood application. In theseembodiments, the amount of blood fluid to be assayed is controlled bythe capacity of assay chamber 107 or the matrix 106 contained therein asdescribed below.

[0084] As described herein for performing blood glucose assays (and asunderstood in the art that essentially the same microfluidics structurescan be used for a multiplicity of blood analyte assays or, moregenerally, for analyte assays in any fluid sample, most preferably abiological fluid sample), a capillary barrier prevents movement of thefluid sample directly into the assay chamber 107. In the meteringstructure shown in FIG. 1A, fluid (or more properly for thisillustrative example, blood) metering capillary 102 acted as a capillarybarrier that prevented blood fluid flow from metering capillary 102 at afirst, non-zero rotational speed f₁, ranging from about 200 rpm to about450 rpm and sufficient to permit fluid flow comprising overflow from theentry port 101 through overflow capillary 121 and into overflow chamber122. This capillary boundary was constructed to be overcome at a secondrotational speed f₂, ranging from about 250 rpm to about 900 rpm (wheref₂>f₁). In the alternative embodiments shown in FIGS. 1B and 1C, bloodfluid chamber 104 acted as a capillary barrier that was maintainedduring rotation at a rotational speed sufficient to motivated excessfluid sample from the entry port 101 to the overflow chamber 122, andwas overcome at a second rotational speed greater than the firstrotational speed to permit fluid sample flow into assay chamber 107.

[0085] Blood metering capillary 102 and blood fluid chamber 104 were indifferent alternatives of the microsystems platforms of the inventionfluidly connected to capillary 110 that was from about 0.02 mm to about2 mm deep and had a cross-sectional diameter of from about 0.02 mm toabout 2 mm and was connected to capillary or sacrificial valve 111.Sacrificial or capillary valve 111 was further fluidly connected withcapillary 112 that was from about 0.02 mm to about 2 mm deep and had across-sectional diameter of from about 0.02 mm to about 2 mm, andfurther to assay chamber 107. In capillary valve embodiments, thejunction between capillary 110 and capillary 112 creates capillary valve111, wherein said capillary valve 111 was from about 0.03 mm to about2.2 mm deep and had a cross-sectional diameter of from about 0.03 mm toabout 2.2 mm. In order to function as a capillary valve, the junctionbetween capillary 110 and capillary 112 must have a depth and/or crosssectional area greater than that of capillary 110 in a disc fabricatedfrom hydrophilic materials such as acrylic. In sacrificial valveembodiments, intermediate melting temperature materials (including, forexample, waxes as described above) are placed in the lumen of capillary110 forming a fluid-tight seal. In these embodiments, chamber 111 isfluidly connected to the sacrificial valve so that melted wax from therelease sacrificial valve is sequestered in the chamber.

[0086] Rectangular assay chamber 107 was constructed in the surface ofthe platform to have a depth of from about 0.2 mm to about 3 mm, mostpreferably comprising a circular or rectangular depression 113 connectedto capillary 112. Depression 113 was constructed to have a volumetriccapacity of from half to twice the assay volume. Assay chamber 107 alsocomprised a pad or matrix 106 of a hydrophilic substance. Materials usedto prepare said matrices include but are not limited to derivatizednylons, nitrocellulose, fiberglass and polyesters, most preferablyhaving a pore size of 0.2-2.0 μm, most preferably comprising apositively-charged nylon matrix having a pore size of about 0.8 μm. Theupper limit on pore size of matrix 106 is chosen to inhibit or preventblood cell entry into the matrix. The matrix is positioned in assaychamber 107 to be in fluidic contact with depression 113, morepreferably covering depression 113, and most preferably having a surfacearea greater than the surface area of depression 113. The matrix wasfurther impregnated with immobilized reagents which produce a detectableproduct proportional to the amount or concentration of glucose in ablood sample. Most preferably, the detectable product is a coloredproduct, i.e., a product absorbing light at a detectable, mostpreferably a visible, wavelength.

[0087] In preparing matrices according to the invention, reagentsadvantageously used to detect and more preferably quantitate an amountof a component of a biological fluid sample are impreganted into thematrix. As a non-limiting example, glucose is detected according to theinvention using a glucose oxidase assays system, as described inadditional detail below. Matrices for performing such assays using themicrofluidics platforms of the invention are prepared by saturating thematrix membrane with an 8 mL solution of distilled water containing 0.12g 2,5-ferandione polymer with methoxylene (CAS: 9011-16-9), 10 mg EDTA,200 mg Polypep® Low Viscosity (Sigma Chemical Company, St. Louis, Mo.),668 mg sodium citrate, 28.75 mg glucose oxidase and 27.3 mg peroxidase.The saturated membrane is then dried and then saturated with 5 mLacetonitrile and 5 mL distilled water containing 40 mg3-methyl-2-benzothiazolinone hydrazone and 80 mg 3-dimethylaminobenzoicacid. The saturated membrane is dried and applied to the microfluidicsdisk of the invention for use.

[0088] In an alternative embodiment shown in FIG. 15, assay chamber 107is comprised of a rectangular cavity in the surface of the platformhaving a depth of from about 0.2 mm to about 3 mm which is fluidlyconnected at its end proximal to the axis of rotation to capillary 112and to air displacement channels 114 and ports 115. The second member ofthe assay chamber is a rectangular piece made from the same material asthe platform or other material and designed to snap into the cavityforming liquid-tight seals around all edges. The snap-in piece has twofaces, an A face and a B face. The A face consists of a fluid entrychannel 112 connected to depression 113; depression 113 is furtherconnected to air displacement channels 114. Depression 113 is from about0.05 mm to about 5 mm deep and has a dimensions from about 0.5 mm by 4mm, having a volumetric capacity of from about half to about twice theassay volume applied to the disc. A pad or matrix 106 is attached to theA face of the snap-in piece, comprising a hydrophilic substancepossessing a pore size of 0.2-2.0 μm, most preferably comprising apositively-charged nylon matrix having a pore size of about 0.8 μm. Theupper limit on pore size of matrix 106 is chosen to inhibit or preventblood cell entry into the matrix.. The matrix is positioned in assaychamber 107 to be in fluidic contact with depression 113, morepreferably covering depression 113, and most preferably having a surfacearea greater than the surface area of depression 113. The matrix isfurther impregnated with immobilized reagents which produce a detectableproduct proportional to the amount or concentration of glucose in ablood sample. Most preferably, the detectable product is a coloredproduct, i.e., a product absorbing light at a detectable, mostpreferably a visible, wavelength.

[0089] As illustrated in FIGS. 2A through 2F, in the use of thisplatform an imprecise volume (ranging from 20-150 μL) of blood wasapplied to the entry port 101. In embodiments of the platform comprisingair displacement channels, the fluid wicked into air channel 114 and wasstopped by capillary junction 115. Fluid also wicked into meteringcapillary 102 and overflow capillary 121. Fluid flowed through themetering capillary 102 and overflow capillary 121 at no rotational speeduntil the fluid reached capillary junctions at the junction betweenmetering capillary 102 and capillary 110 and overflow capillary 121 andoverflow chamber 122. Metering capillary 102 was constructed to define aprecise volume from about 15 to about 60 μL of fluid between entry port101 and capillary junction 111, which was designed to be at least theamount of the fluid placed by the user in entry port 101.

[0090] After sample loading by a user and filling of metering capillary102 and overflow capillary 121 at no rotational speed, the platform wasspun at a first rotational speed f₁, ranging from about 50 rpm to about600 rpm, which was sufficient to motivate fluid flow through theoverflow capillary 121 in this microfluidics array having an entry port101 with a depth of from about 0.2 mm to about 3 mm, metering capillary102 with dimensions of about 0.5 mm×0.5 mm in cross-section and a lengthof about 2.2-3.8 cm from the center of rotation and an overflowcapillary 121 with dimensions of about 0.5 mm×0.5 mm in cross-sectionand a length of about 5.4 cm from the center of rotation.

[0091] Due to the greater distance of the end of overflow capillary 121from the center of rotation than the end of metering capillary 102, atrotational speed f₁ fluid flowed through overflow capillary 121 intooverflow chamber 122. The platform was spun until all excess fluid isevacuated from entry port 101 and into overflow chamber 122, except thefluid contained in metering chamber 102.

[0092] At a second rotational speed f₂ of from about 100 rpm to about1000 rpm, the precise amount of fluid contained in metering capillary102 was delivered into assay chamber 107. In embodiments comprising asacrificial valve 111 in-line with capillary 110 at a position betweencapillary 110 and 112 shown in FIG. 2B, release of the sacrificial valveresulted in fluid flow through capillary 112 and into assay chamber 107.In said embodiments, fluid flow is achieved at rotational speed f₂ withremoval of the sacrificial valve. In embodiments of the platforms of theinvention comprising capillary valve 111 at a position between capillary110 and 112 shown in FIG. 2B, capillary 110 preferably filled along withfilling of metering capillary 102 until blood reached capillary junction111 at the junction between capillary 110 and capillary 112; in suchembodiments, the capillary junction had a depth of from about 0.03 mm toabout 2.2 mm, or at least greater than the depth of capillary 110.

[0093] Blood flowing into assay chamber 107 is preferentially directedto depression 113 in the assay chamber; the dimensions of depression 113are conveniently chosen to be able to contain substantially all of theblood fluid of the sample metered through metering capillary 102 intoassay chamber 107 (FIG. 2C). Displaced air flows through air channel114, and may be vented to the surface of the disc or in communicationwith blood fluid chamber 104.

[0094] As blood flows into depression 113, the fluid component of theblood is driven by pressure and hydrophilic forces into matrix 106; thepore size of the matrix is chosen to prevent the cellular components ofthe blood from entering the matrix (FIG. 1.4). In preferred embodiment,the cellular component of the blood is retained in depression 113 andthe fluid component is efficiently distributed by wicking and bycentripetal force into matrix 106. In an alternative embodiment,alternative matrix 118 comprises at least two distinct elements that arecompressed or adhered to one another in the assays chamber. The firstelement is similar to the reagent-containing matrix 106 described above;however, this embodiment of the matrix has a pore size that is notlimited by the size of cellular components of the blood, and can be anypore size deemed optimal on experimental, economic, manufacturing oravailability grounds. The second element comprises a filtering layerhaving a pore size that prevents cells and cellular debris from enteringthis portion of the matrix. In a preferred embodiment, the two elementsand rearranged in assay chamber 107 so that second matrix element is incontact with depression 113 wherein the blood aliquot is first contactedwith the second matrix component. Blood fluid flows into and through thesecond matrix element and into the first matrix element, wherebycellular components of blood are prevented from entering assay chamber107 by the pore size of second matrix element. Preferably, thedimensions of the matrix element is about 1 cm by about 0.75 cm and hasa thickness of about 0.05 cm.

[0095] As the fluid component of the blood wicks into matrix 106, driedreagents are solubilized and the reaction of the blood componentcatalyzed by said reagents proceeds. The timescale over which thesechemical reactions take place is chosen to be long compared with thetime it takes for the fluid component of blood to saturate the matrix106. The time it takes for the blood fluid to saturate the matrix isdependent on the capacity of the matrix 106 to absorb the fluid and thedelivery speed of the blood fluid to the assay chamber 107, which inturn is dependent on the rotational velocity and the cross-sectionaldimensions of capillary 110. In preferred embodiments, the reaction(s)goes to completion within about 1 min. Reaction of the bloodcomponent(s) with the reagents produce colored product which is thendetected (FIG. 2E). In preferred embodiments, detection is performedspectrophotometrically, including absorbance, transmittance, reflection,fluorescence, and chemiluminescence, although visual inspection is alsocontemplated in alternative embodiments of the invention.

[0096] In an alternative embodiment, assay chamber 107 further comprisesa detection cell 117 that is laterally adjacent to the portion of assaychamber 107 comprising depression 113 (shown in FIGS. 3A through 3E).This embodiment uses a variation of alternative matrix 118 wherein thematrix comprises a blood separation element and a blood fluid wickingelement. These elements are arranged in assay chamber 107 so that theblood separation element is in contact with depression 113 and the bloodfluid wicking element is compressed or attached to the blood separationelement along its entire extent. A blood fluid wicking element havingdimensions as described above and sufficient to encompass the entiresurface extent of the blood separation element and to further extendthroughout the complete extent of assay chamber 107, including detectioncell 117. Reagents are only present in the portion of the blood fluidwicking element in that portion of the element comprising detection cell117; this arrangement is shown in detail in FIG. 3A.

[0097] In the practice of this embodiment of the invention, blooddelivered to depression 113 wicks through the blood separation elementand into the blood fluid wicking element (FIGS. 3B and 3C). As theregion of blood fluid wicking element above depression 113 saturateswith blood plasma, the blood fluid wicks laterally into the portion ofthe matrix 118 comprising detection cell 117 (FIG. 3D). Wetting of thisportion of the matrix, which comprises the immobilized reagents andinitiates the glucose detection reaction(s) as described in Example 1,with the production of a colored product (FIG. 3E). The amount ofcolored product produced is detected and the amount of glucose in theblood sample determined thereby.

[0098] Another alternative embodiment of a blood glucose assayMicrosystems platform is shown in FIGS. 4A through 4E. Construction ofthe disk embodiments of the platforms of the invention were as describedabove.

[0099] The blood application and metering components and theirdimensions and relationships to one another are identical to thosedescribed above, comprising sample entry port chamber 201, meteringcapillary 202, overflow capillary 203, and overflow chamber 205. As inExample 1, each of the overflow and fluid chambers is also connectedwith air ports or air channels, such as 214, and capillary junction(s)215, that permit venting of air displaced by fluid movement on theplatform.

[0100] Metering capillary 202 is fluidly connected to capillary 210 thatwas from about 0.02 mm to about 2 mm deep and has a cross-sectionaldiameter of from about 0.02 mm to about 2 mm and is connected tocapillary or sacrificial valve 211. Sacrificial or capillary valve 211is further fluidly connected with capillary 212 that is from about 0.02mm to about 2 mm deep and has a cross-sectional diameter of from about0.02 mm to about 2 mm; capillary 212 is further fluidly connected toassay chamber 207. Where capillary 210 is connected with capillary valve211, said capillary valve 211 is from about 0.03 mm to about 2.2 mm deepand has a cross-sectional diameter of from about 0.03 mm to about 2.2mm.

[0101] Assay chamber 207 comprises a depression in the surface of theplatform having a depth of from about 0.2 mm to about 3 mm preferablycomprising a circular or rectangular depression 213 connected tocapillary 212 and most preferably wherein a metered or otherwisecontrolled amount of blood can be contained in the assay chamber. Assaychamber 207 also comprises a pad or matrix 206 of a hydrophilicsubstance possessing a pore size of from about 0.2 to about 2 μm, mostpreferably about 0.8 μm. The upper limit on pore size of matrix 206 ischosen to inhibit or prevent blood cell entry into the matrix, and topromote entry or wicking of the fluid component of blood (plasma orserum) to enter the body of the matrix. The matrix is positioned inassay chamber 207 to be in fluidic contact with depression 213, morepreferably covering depression 213, and most preferably having a surfacearea greater than the surface area of depression 213; in embodiments nothaving depression 213 as a component of assay chamber 207, the matrixsubstantially fills the volumetric extent of the assay chamber. Thematrix is further impregnated with immobilized reagents which produce adetectable product proportional to the amount or concentration ofglucose in a blood sample. Most preferably, the detectable product is acolored product, i.e., a product absorbing light at a detectable, mostpreferably a visible, wavelength.

[0102] In an alternative embodiment, assay chamber 207 comprises asurface wherein reagents 219 are directly dried or otherwise immobilizedon the surface of the chamber, and in such embodiments the assay chamberdoes not comprise matrix 206.

[0103] Assay chamber 206 is further fluidly connected with capillary 220at a position preferably most distal to the axis of rotation. Capillary220 is from about 0.02 mm to about 2 mm deep and has a cross-sectionaldiameter of from about 0.02 mm to about 2 mm, and is further fluidlyconnected with waste reservoir 221. Waste reservoir 221 is positionedfrom about 1.2 cm to about 14 cm from the axis of rotation, and has adepth in the platform of from about 0.2 mm to about 3 mm. Assay chamber206 is also fluidly connected with capillary 222 at a positionpreferably most proximal to the axis of rotation. Capillary 222 is fromabout 0.02 mm to about 2 mm deep and has a cross-sectional diameter offrom about 0.02 mm to about 2 mm, and is further fluidly connected withwash buffer reservoir 223. Wash buffer reservoir 223 has a depth in theplatform of from about 0.2 mm to about 3 mm and is positioned from about1.2 cm to about 14 cm from the axis of rotation, and in any event moreproximal to the axis than assay chamber 206. Fluid flow from wash bufferreservoir 223 through capillary 222 is controlled by either capillaryvalve 224 or sacrificial valve 225. Where capillary 222 was connectedwith capillary valve 224, said capillary valve 224 is from about 0.03 mmto about 2.2 mm deep and has a cross-sectional diameter of from about0.03 mm to about 2.2 mm.

[0104] As illustrated in FIGS. 4A through 4D, in the use of thisplatform an imprecise volume (ranging from 20-150 μL of fluid) of bloodis applied to the entry port 201. Application of blood to the platformand delivering a metered amount of blood to metering capillary 202 wasachieved as described above. Alternatively, an unmetered amount of bloodis introduced onto the platform directly into assay chamber 207.Preferably, from about 15 μL to about 150 μL of blood is delivered toassay chamber 207.

[0105] At a rotational speed f₂ of about 100-1000 rpm, the preciseamount of fluid contained in metering capillary 202 is delivered intoassay chamber 207. In embodiments comprising a sacrificial valve 211in-line with capillary 210 at a position between capillary 210 and assaychamber 207 as shown in FIG. 3.1, release of the sacrificial valveresults in fluid flow through capillary 212 and into assay chamber 207.In said embodiments, fluid flow is achieved at rotational speed f₂ withremoval of the sacrificial valve. In embodiments of the platforms of theinvention comprising capillary valve 211 at a position between capillary210 and 212 as shown in FIG. 3B, capillary 210 preferably fills alongwith filling of blood metering capillary 202 until blood reachescapillary junction 211 at the junction between capillary 210 andcapillary 212; in such embodiments, the capillary junction had a depthof from about 0.03 mm to about 2.2 mm. At a rotational speed f₂ of about100-1000 rpm, the fluid contained in blood metering capillary 202 isdelivered into assay chamber 207 (FIG. 3C).

[0106] Blood flowing into assay chamber 207 is directed to matrix 206 inthe assay chamber; the dimensions of matrix 206 are conveniently chosento be able to contain substantially all of the blood fluid of the samplemetered through metering capillary 202 into assay chamber 207. Displacedair flows through air channel 214, and may be vented to the surface ofthe disc or in communication with blood metering capillary 202 or entryport 201. Alternatively, in embodiments wherein the blood sample isunmetered, the capacity of the matrix 206 is sufficient to absorb acontrolled amount of blood. In further alternative embodiments, whereinassay chamber does not comprise matrix 206, the assay chamber isprovided having a capacity for a controlled volume of blood.

[0107] As blood flows into assay chamber 207, the fluid component of theblood wicks into matrix 206; the pore size of the matrix is chosen toprevent the cellular components of the blood from entering the matrix.In preferred embodiment, the cellular component of the blood is retainedin assay chamber 207 and the fluid component is efficiently wickeduniformly throughout matrix 206.

[0108] As the fluid component of the blood wicks into matrix 206, driedreagents are solubilized and the reaction of the blood componentcatalyzed by said reagents proceeds. The timescale over which thesereactions take place is chosen to be long compared with the time ittakes for the fluid component of blood to saturate the matrix 206;however, in preferred embodiments, the reaction(s) goes to completionwithin about 0.5 to about 5 min. Reaction of the blood component(s) withreagents produce colored product which is then detected. In preferredembodiments, detection is performed spectrophotometrically, althoughvisual inspection is also contemplated in alternative embodiments of theinvention.

[0109] After a time sufficient to produce a detectable amount of acolored product, the wash buffer is released from wash buffer reservoir223 through capillary 222 and into assay chamber 207. In embodimentscomprising a sacrificial valve 225 in-line with capillary 222 at aposition between capillary 222 and assay chamber 207 shown in FIG. 3.3,release of the sacrificial valve results in fluid flow through capillary222 and into assay chamber 207. In said embodiments, fluid flow isachieved at rotational speed f₃ of about 250 rpm to about 1500 rpm withremoval of the sacrificial valve. In embodiments of the platforms of theinvention comprising capillary valve 224 at a position between capillary222 and assay chamber 207 shown in FIG. 3D, capillary 222 preferablyfills along with filling of blood fluid chamber 204 until blood reachedcapillary junction 224; in such embodiments, the capillary junction hada depth of from about 0.03 mm to about 2.2 mm. At a higher rotationalspeed f₄ of about 400-2000 rpm, the fluid contained in wash reservoir223 is delivered into assay chamber 207 (FIG. 3D). Because the fluidflow of wash buffer into the assay chamber and fluid from the assaychamber to the waste chamber is laminar, there is very little mixing ofthe washing fluid with the fluid initially in the assay chamber. Thewash fluid displaces the fluid sample in the assay by pushing it intothe waste chamber. The exit of capillary 220 into chamber 221 is at aradial position such that assay chamber 207 must remain filled withfluid during this washing process. The quality of fluid removal is suchthat no more than 1 part in 1000 of the fluid in the chamber 207 (whichhas not been imbibed into matrix 206) remains. The fluid which haswicked into matrix 206 is not removed during this wash because of thesmall pore size of the matrix which resists fluid flow; furthermore,color reagents do not diffuse out of matrix 206 if the wash time isrelatively short (less than a few hundred seconds). As a result,interfering blood fluid components such as hemoglobin are removed fromchamber 207 while substantially leaving behind color reagents in matrix206. These are then interrogated spectrophotometrically in assay chamber207 (FIG. 4E).

[0110] Another alternative embodiment of a blood glucose assayMicrosystems platform is shown in FIGS. 5A through 5E. Construction ofthe disc embodiments of the platforms of the invention were as describedabove.

[0111] The blood application and metering components and theirdimensions and relationships to one another are identical to thosedescribed above, comprising sample entry port chamber 301, meteringcapillary 302, overflow capillary 303, and overflow chamber 305. As inExample 1, each of the overflow and fluid chambers is also connectedwith air ports or air channels, such as 314, and capillary junction(s)315, that permit venting of air displaced by fluid movement on theplatform.

[0112] Metering capillary 302 is fluidly connected to capillary 310 thatis from about 0.02 mm to about 2 mm deep and has a cross-sectionaldiameter of from about 0.02 mm to about 2 mm and is connected tocapillary or sacrificial valve 311. Sacrificial or capillary valve 311is further fluidly connected with capillary 312 that is from about 0.02mm to about 2 mm deep and has a cross-sectional diameter of from about0.02 mm to about 2 mm, and is further fluidly connected to assay chamber307. Where capillary 310 is connected with capillary valve 311, saidcapillary valve 311 is from about 0.03 mm to about 2.2 mm deep and has across-sectional diameter of from about 0.03 mm to about 2.2 mm.

[0113] Assay chamber 307 comprises a pad or matrix 306 of a hydrophilicsubstance possessing a pore size of from about 0.2 μm to about 2 μm,most preferably about 0.8 μm. The upper limit on pore size of matrix 306is chosen to inhibit or prevent blood cell entry into the matrix. Thematrix is further impregnated with immobilized reagents which produce adetectable product proportional to the amount or concentration ofglucose in a blood sample. Most preferably, the detectable product is acolored product, i.e., a product absorbing light at a detectable, mostpreferably a visible, wavelength.

[0114] As illustrated in FIGS. 5A through 5E, in the use of thisplatform an imprecise volume (ranging from about 15 μL to about 150 μLof fluid) of blood is applied to the entry port 301. Application ofblood to the platform and delivering a metered amount of blood to bloodmetering capillary 302 was achieved as described above. Alternatively,an unmetered amount of blood is introduced onto the platform directlyinto assay chamber 307, preferably, from about 15 μL to about 150 μL.

[0115] At a rotational speed f₁ of 100-1000 rpm the precise amount offluid contained in metering chamber 302 is delivered into assay chamber307. In embodiments comprising a sacrificial valve 311 in-line withcapillary 310 at a position between capillary 310 and 312 shown in FIG.5A, release of the sacrificial valve results in fluid flow throughcapillary 312 and into assay chamber 307. In said embodiments, fluidflow is achieved at rotational speed f₁ with removal of the sacrificialvalve. In embodiments of the platforms of the invention comprisingcapillary valve 311 at a position between capillary 310 and 312 shown inFIG. 5B, capillary 310 preferably fills along with filling of bloodmetering capillary 302 until blood reaches capillary junction 311 at thejunction between capillary 310 and capillary 312; in such embodiments,the capillary junction has a depth of from about 0.03 mm to about 2.2mm. At a higher rotational speed f₂ of about 250 rpm to about 1500 rpm,the fluid contained in blood metering capillary 302 is delivered intoassay chamber 307 (FIG. 5C).

[0116] Blood flows into assay chamber 307 with displaced air flowingthrough air channel 314, and may be vented to the surface of the disc orin communication with blood metering capillary 302 or entry port 301(FIG. 5C). As blood flows into assay chamber 307, the fluid component ofthe blood wicks into matrix 306; the pore size of the matrix is chosento prevent the cellular components of the blood from entering thematrix. In an alternative embodiment, alternative matrix 318 comprisesat least two distinct elements that are compressed or adhered to oneanother in the assays chamber. The first element is similar to thereagent-containing matrix 306 described above; however, this embodimentof the matrix has a pore size that is not limited by the size ofcellular components of the blood, and can be any pore size deemedoptimal on experimental, economic, manufacturing or availabilitygrounds. The general dimensions of the matrix are equivalent to thedimensions disclosed above, so that the matrices are advantageouslystandardized and interchangeable on the microsystems platforms of theinvention. The second element comprises a filtering layer having a poresize that prevents cellular components from entering this portion of thematrix. In a preferred embodiment, these elements are arranged in assaychamber 307 so that the second matrix element is in contact with thesurface of assay chamber 307 wherein the blood aliquot is firstcontacted with the second matrix component. Blood fluid wicks into andthrough second matrix element and into the first matrix element, wherebycellular components of blood are prevented from entering assay chamber307 by the pore size of second matrix element.

[0117] As the fluid component of the blood wicks into matrix 306, driedreagents are solubilized and the reaction of the blood componentcatalyzed by said reagents proceeds as described below. The timescaleover which these reactions take place is chosen to be long compared withthe time it takes for the fluid component of blood to saturate thematrix 306; however, in preferred embodiments, the reaction(s) goes tocompletion within about 0.5 to about 5 min. Reaction of the bloodcomponent(s) with reagents produce colored product (FIG. 5D). Aftersufficient time for the reaction to proceed, the platform is centrifugedat a speed of from about 800 rpm to about 200 rpm, wherein said speedpellets the cellular component of the blood, particularly the red bloodcell component thereof, to the “bottom” or most axially distal portionof assay chamber 307. Detection of the colored product of the glucosedetecting reaction in matrix 306 is performed at a position in assaychamber 307 radially more proximal to the axis of rotation than theposition to which the cellular fraction has been pelleted (FIG. 5E). Inpreferred embodiments, detection is performed spectrophotometrically,although visual inspection is also contemplated in alternativeembodiments of the invention. The amount of colored product produced isdetected and the amount of glucose in the blood sample determinedthereby.

[0118] Another alternative embodiment of a blood glucose assayMicrosystems platform is shown in FIGS. 6A through 6E. Construction ofthe disk embodiments of the platforms of the invention were as describedabove.

[0119] The blood application and metering components and theirdimensions and relationships to one another are identical to thosedescribed above, comprising sample entry port chamber 401, meteringcapillary 402, overflow capillary 403, and overflow chamber 405. As inExample 1, each of the overflow and fluid chambers is also connectedwith air ports or air channels, such as 414, and capillary junction(s)415, that permit venting of air displaced by fluid movement on theplatform.

[0120] Metering capillary 402 is fluidly connected to capillary 410 thatis from about 0.02 mm to about 2 mm deep and has a cross-sectionaldiameter of from about 0.02 mm to about 2 mm and is connected tocapillary or sacrificial valve 411. Sacrificial or capillary valve 411is further fluidly connected with capillary 412 that is from about 0.02mm to about 2 mm deep and has a cross-sectional diameter of from about0.02 mm to about 2 mm. Capillary 412 is further fluidly connected tocell separation chamber 407. Where capillary 410 is connected withcapillary valve 411, said capillary valve 411 is from about 0.02 mm toabout 2 mm deep and has a cross-sectional diameter of from about 0.02 mmto about 2 mm.

[0121] Cell separation chamber 407 comprised a depression in the surfaceof the platform having a depth of from about 0.02 mm to about 3 cm, mostpreferably comprising a circular or rectangular depression 413 connectedto capillary 412. Cell separation chamber also comprises a filter 406consisting of a porous material whose pores are sized (from about 0.2 μmto about 2 μm) to filter red blood cells. Filter 406 is in contact withand more preferably adhered to the surface of cell separation chamber407 and most extends over depression 413. Blood flowing into cellseparation chamber 407 is directed to depression 413 by capillary 412.As depression 413 fills, serum is both wicked and driven byrotation-induced pressure through the filter 406 to the upper surface ofcell separation chamber 407, leaving red blood cells and other cellularcomponents trapped beneath filter 406 in depression 413. The volume ofblood fluid at the upper surface of cell separation chamber 407 rangesfrom about 15 μL to about 150 μL.

[0122] Cell separation chamber 407 is fluidly connected at a radialposition distal from the axis of rotation to capillary 420 that is fromabout 0.02 mm to about 2 mm deep and has a cross-sectional diameter offrom about 0.02 mm to about 2 mm. Capillary 420 is further fluidlyconnected with assay chamber 421 that is from about 0.02 mm to about 3cm deep and has a cross-sectional diameter of from about 0.02 mm toabout 10 cm. Assay chamber 421 comprises a pad or matrix 422 of ahydrophilic substance possessing any convenient pore size, for example,a pore size of 0.2-2.0 μm, such as a pore size of about 0.8 μm. Thematrix is further impregnated with immobilized reagents which produce adetectable product proportional to the amount or concentration ofglucose in a blood sample. Most preferably, the detectable product is acolored product, i.e., a product absorbing light at a detectable, mostpreferably a visible, wavelength.

[0123] As illustrated in FIGS. 6A through 6E, in the use of thisplatform an imprecise volume (ranging from 15 to about 150 μL of fluid)of blood is applied to the entry port 401. Application of blood to theplatform and delivering a metered amount of blood to metering capillary402 was achieved as described in Example 1 above. At a rotational speedf₁ of 100-1000 rpm, the precise amount of fluid contained in meteringcapillary 402 is delivered into assay chamber 407. Alternatively, anunmetered amount of blood is introduced onto the platform directly intoassay chamber 407, preferably, from about 15 μL to about 150 μL.

[0124] Fluid movement into metering capillary 402 is accompanied byfilling of capillary 410. In embodiments comprising a sacrificial valve411 in-line with capillary 410 at a position between capillary 410 and412 shown in FIG. 6A, release of the sacrificial valve results in bloodfluid flow through capillary 412 and into cell separation chamber 407.In said embodiments, fluid flow is achieved at rotational speed f₁ withremoval of the sacrificial valve. In embodiments of the platforms of theinvention comprising capillary valve 411 at a position between capillary410 and 412 shown in FIG. 6A, capillary 410 preferably fills along withfilling of metering capillary 402 until blood reaches capillary junction411 at the junction between capillary 410 and capillary 412. In suchembodiments, the capillary junction has a depth of from about 0.03 mm toabout 2.2 mm. In these embodiments, the fluid contained in meteringcapillary 402 is delivered into cell separation chamber 407 by rotationof the disc at a higher rotational speed f₂ of from about 250 rpm toabout 1500 rpm (FIG. 6B).

[0125] Blood flows into cell separation chamber 407 with displaced airflowing through air channel 414, and may be vented to the surface of thedisc or in communication with metering capillary 402 or entry port 401(FIG. 6C). As blood flows into cell separation chamber 407, the fluidcomponent of the blood wicks into matrix 406; the pore size of thematrix is chosen to prevent the cellular components of the blood fromentering the matrix. In a preferred embodiment, matrix 406 is arrangedin cell separation chamber 407 so that the matrix is in contact with ormore preferably adhered to the lower surface of cell separation chamber407. Blood fluid, such as plasma or serum, traverses matrix 406 bywicking and under rotation-induced pressure, saturating the matrix andfilling a space formed between the top surface of the matrix and the topsurface of cell separation chamber 407 (FIG. 6C).

[0126] Blood fluid exits cell separation chamber 407 through capillary420 and flows into assay chamber 421 (FIG. 6D), with displaced airflowing through air channel 414, and may be vented to the surface of thedisc or in communication with cell separation chamber 407 (FIG. 5.4).Blood fluid flows into assay chamber 421 and wicks into matrix 422. Asthe blood fluid wicks into matrix 422, dried reagents are solubilizedand the reaction of the blood component catalyzed by said reagentsproceeds as described below. The timescale over which these reactionstake place is chosen to be long compared with the time it takes for thefluid component of blood to saturate the matrix 422; however, inpreferred embodiments, the reaction(s) goes to completion within about0.5 min to about 5 min. Reaction of the blood component(s) with reagentsproduce a colored product (FIG. 6E). In preferred embodiments, detectionof colored product is performed spectrophotometrically, although visualinspection is also contemplated in alternative embodiments of theinvention. The amount of colored product produced is detected and theamount of glucose in the blood sample determined thereby.

[0127] Another alternative embodiment of a blood glucose assaymicrosystems platform is shown in FIGS. 7A-7D. Construction of the diskembodiments of the platforms of the invention were as described above.

[0128] The blood application and metering components and theirdimensions and relationships to one another are identical to thosedescribed above, comprising sample entry port chamber 501, meteringcapillary 502, overflow capillary 503, and overflow chamber 505. As inExample 1, each of the overflow and fluid chambers is also connectedwith air ports or air channels, such as 514, and capillary junction(s)515, that permit venting of air displaced by fluid movement on theplatform.

[0129] Metering capillary 502 is fluidly connected with capillary 510,having a cross-sectional diameter of from about 0.02 mm to about 2 mmand extending from about 1 mm to about 5 cm from the blood fluidchamber. Capillary 510 is also fluidly connected with mixing chamber 515that is from about 0.02 mm to about 3 cm deep and having across-sectional diameter of from about 0.02 mm to about 10 cm, and ispositioned from about 1.2 cm to about 14 cm from the center of rotation.Mixing chamber 515 is also fluidly connected with capillary 520 having across-sectional diameter of from about 0.02 mm to about 2 mm. Capillary520 is further fluidly connected with reagent chamber 521, whereinreagents 508 for detecting blood glucose and determining theconcentration thereof are stored. Reagent chamber 521 is from about 0.02mm to about 3 cm deep and having a cross-sectional diameter of fromabout 0.02 mm to about 10 cm, and is positioned from about 1.2 cm toabout 14 cm from the center of rotation. In certain embodiments,reagents 508 are stored on the disc in solution; in these embodiments,fluid flow through capillary 520 is preferably controlled usingsacrificial valve 525. In alternative embodiments, reagents 508 arestored in reagent chamber 521 in dry form. In these embodiments,capillary 520 can preferably comprise sacrificial valve 525 or capillaryvalve 525 positioned between mixing chamber 515 and reagent chamber 521.In these embodiments, reagent chamber 521 further comprises means for auser to add an appropriate amount of a reagent diluent 530, or theplatform further comprises diluent chamber 531 fluidly connected toreagent chamber 521 by way of capillary 532. In these embodiments,reagent diluent chamber 531 that is from about 0.02 mm to about 3 cmdeep and having a cross-sectional diameter of from about 0.02 mm toabout 10 cm, and is positioned from about 1.2 cm to about 14 cm from thecenter of rotation. In these embodiments, reagents 508 are solubilizedin reagent chamber 521 immediately prior to or during use.

[0130] Mixing chamber 515 is fluidly connected by capillary channel 536having a cross-sectional diameter of from about 0.02 mm to about 2 mmand extending from about 1 mm to about 5 cm from the mixing chamber.Capillary channel 536 is further fluidly connected with mixed fluidreceiving chamber 537. Mixed fluid chamber 537 is from about 0.02 mm toabout 3 cm deep and having a cross-sectional diameter of from about 0.02mm to about 10 cm, and is positioned from about 1.2 cm to about 14 cmfrom the center of rotation. Alternatively, capillary 536 is furtherfluidly connected to second mixing chamber 540. Second mixing chamber540 is from about 0.02 mm to about 3 cm deep and having across-sectional diameter of from about 0.02 mm to about 10 cm, and ispositioned from about 1.2 cm to about 14 cm from the center of rotation.Second mixing chamber is fluidly connected by a capillary channel 546which is further connected with mixed fluid receiving chamber 537. Inthese embodiments, capillary channel 546 has a cross-sectional diameterof from about 0.02 mm to about 2 mm and extends from about 1 mm to about5 cm from the second mixing chamber.

[0131] Capillary channels 510 and 520, and capillary channels 536 and546, may be offset in their connection with the mixing chamber(s). As aconsequence, fluid flowing through capillary channels 536 and 546 isforced to encounter the opposite wall of mixing chambers 515 and 540before fluid flow can proceed through further capillary channels. Thefluid streams entering a small channel flow in a laminar fashion andtherefore mix only by diffusion; the mixing chamber allows the fluids tomove in a turbulent fashion and thus mix more effectively. This resultsin the creation of turbulence in the mixed laminar fluid stream incapillary channels 536 and 546 caused by the conjoint flow of fluid fromthe input capillaries without appreciable mixing. The turbulence createdby the structure of mixing chambers 515 and 540 is sufficient to disruptlaminar flow and cause fluid mixing in the chamber prior to continuedfluid flow through capillary channel 546 and into mixed fluid receivingchamber 537.

[0132] As illustrated in FIGS. 7A through 7D, in the use of thisplatform a volume of blood is applied to metering capillary 502, eitherdirectly or using the metering components of the platform describedabove. Fluid enters the each of the capillaries 510 and 520 and stops atcapillary junction(s) or sacrificial valve(s) 525.

[0133] At a rotational speed f₁ of 100 to 1000 rpm, the fluids from eachcapillary flow past capillary junction 525 and through mixing chamber515 (FIG. 7B). Alternatively, fluid flow is activated by release ofsacrificial valves 525. Fluid flow within mixing chamber 515 isturbulent, in contrast to fluid flow through capillaries 510 and 520,which is primarily laminar, so that mixing occurs predominantly inmixing chamber 515. Fluid flow proceeds through channel 536 and theneither through second mixing chamber 540 or directly through capillary546 into mixed fluid receiving chamber 537 (FIG. 7C).

[0134] Glucose detection reagents mixed with blood reacts in mixed fluidreceiving chamber 537 (FIG. 7C). The timescale over which thesereactions take place preferably goes to completion within about 0.5 minto about 5 min. Reaction of the blood component(s) with reagents 508produce a colored product (FIG. 7D). After sufficient time for thereaction to proceed, the platform is centrifuged at a speed of about 500rpm to about 3000 rpm, wherein said speed pellets the cellular componentof the blood, particularly the red blood cell component thereof, to the“bottom” or most axially distal portion of mixed fluid receiving chamber537. Detection of the colored product of the glucose detecting reactionis performed at a position in mixed fluid receiving chamber 537 radiallymore proximal to the axis of rotation than the position to which thecellular fraction has been pelleted (FIG. 7D). In preferred embodiments,detection is performed spectrophotometrically, although visualinspection is also contemplated in alternative embodiments of theinvention. The amount of colored product produced is detected and theamount of glucose in the blood sample determined thereby.

[0135] Another alternative embodiment of a blood glucose assayMicrosystems platform is shown in FIGS. 8A through 8D. Construction ofthe disk embodiments of the platforms of the invention were as describedabove.

[0136] The blood application and metering components and theirdimensions and relationships to one another are identical to thosedescribed above, comprising sample entry port chamber 601, meteringcapillary 602, overflow capillary 603, and overflow chamber 605. As inExample 1, each of the overflow and fluid chambers is also connectedwith air ports or air channels, such as 634, and capillary junction(s)635, that permit venting of air displaced by fluid movement on theplatform.

[0137] Metering capillary 602 is fluidly connected to capillary 610 thatis from about 0.02 mm to about 2 mm deep and has a cross-sectionaldiameter of from about 0.02 mm to about 2 mm and is connected tocapillary or sacrificial valve 611. Sacrificial or capillary valve 611is further fluidly connected with capillary 612 that is from about 0.02mm to about 2 mm deep and has a cross-sectional diameter of from about0.02 mm to about 2 mm. Capillary 612 is further fluidly connected tocell separation chamber 607. Cell separation chamber 607 is from about0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about002 mm to about 3 cm, and is positioned from about 1.2 cm to about 14 cmfrom the center of rotation. Where capillary 610 is connected withcapillary valve 611, said capillary valve 611 is from about 0.03 mm toabout 2.2 mm deep and has a cross-sectional diameter of from about 0.03mm to about 2.2 mm.

[0138] Cell separation chamber 607 comprises a depression in the surfaceof the platform having a depth of from about 0.02 mm to about 3 cm, mostpreferably comprising a circular or concave depression 613 connected tocapillary 612. Depression 613 has a depth of from about 0.02 mm to about1 cm and a volume of from 15 μL to about 150 μL. Cell separation chamber607 also comprises a filter 606 consisting of a porous material whosepores are sized to filter red blood cells, ranging from about 0.2 μm toabout 2 μm. Filter 606 is in contact with and more preferably adhered tothe surface of cell separation chamber 607 and most preferably extendsover depression 613. Blood flowing into cell separation chamber 607 isdirected to depression 613 by capillary 612. As depression 613 fills,serum is both wicked and driven by rotation-induced pressure through thefilter 606 to the upper surface of cell separation chamber 607, leavingred blood cells and other cellular components trapped beneath filter 606in depression 613. The volume of blood fluid at the upper surface ofcell separation chamber 607 is from about 15 μL to about 150 μL.

[0139] Cell separation chamber 607 is fluidly connected at a radialposition distal from the axis of rotation to capillary 620 that is fromabout 0.02 mm to about 2 mm deep, has a cross-sectional diameter of fromabout 0.02 mm to about 2 mm and extends from about 1 mm to about 5 cmfrom cell separation chamber 607. Capillary 620 is further fluidlyconnected with mixing chamber 615 that is from about 0.02 mm to about 3cm deep, has a cross-sectional diameter of from about 0.02 mm to about10 cm, and is positioned from about 1.2 cm to about 14 cm from thecenter of rotation. Capillary 620 is further fluidly connected withreagent chamber 621, wherein reagents 608 for detecting blood glucoseand determining the concentration thereof is stored. Reagent chamber 621is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameterof from about 0.02 mm to about 10 cm, and is positioned from about 1.2cm to about 14 cm from the center of rotation. In certain embodiments,reagents 608 are stored on the disc in solution; in these embodiments,fluid flow through capillary 620 is preferably controlled usingsacrificial valve 625. In alternative embodiments, reagents 608 arestored in reagent chamber 621 in dry form. In these embodiments, theplatform further comprises diluent chamber 631 fluidly connected toreagent chamber 621 by way of capillary 632. Diluent chamber 631 is fromabout 0.02 mm to about 3 cm deep, has a cross-sectional diameter of fromabout 0.02 mm to about 10 cm, and is positioned from about 1.2 cm toabout 14 cm from the center of rotation. Capillary 632 has across-sectional diameter of from about 0.02 mm to about 2 mm, and ispositioned from about 1.2 cm to about 14 cm from the center of rotation.In these embodiments, reagents 608 are solubilized in reagent chamber621 immediately prior to or during use.

[0140] Mixing chamber 615 is fluidly connected by capillary channel 636having a cross-sectional diameter of from about 0.02 mm to about 2 mmand extending from about 1 mm to about 5 cm from the mixing chamber.Capillary channel 636 is further fluidly connected with mixed fluidreceiving chamber 637. Mixed fluid chamber 637 is from about 0.02 mm toabout 2 mm deep, has a cross-sectional diameter of from about 0.02 mm toabout 2 mm, and is positioned from about 1.2 cm to about 14 cm from thecenter of rotation.

[0141] Alternatively, capillary 636 is further fluidly connected tosecond mixing chamber 640. Second mixing chamber 640 is from about 0.02mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02mm to about 10 cm, and is positioned from about 1.2 cm to about 10 cmfrom the center of rotation. Second mixing chamber is fluidly connectedby a capillary channel 646 which is further connected with mixed fluidreceiving chamber 637. Capillary channel 646 has a cross-sectionaldiameter of from about 0.02 mm to about2 mm and extends from about 1 mmto about 5 cm from the second mixing chamber.

[0142] Capillary channels 610 and 620, and capillary channels 636 and646, may be offset in their connection with the mixing chamber(s). As aconsequence, fluid flowing through capillary channels 636 and 646 areforced to encounter the opposite wall of mixing chambers 615 and 640before fluid flow can proceed through further capillary channels. Thisresults in the creation of turbulence in the mixed laminar fluid streamin capillary channels 636 and 646 caused by the conjoint flow of fluidfrom the input capillaries without appreciable mixing. The turbulencecreated by the structure of mixing chambers 615 and 640 is sufficient todisrupt laminar flow and cause fluid mixing in the chamber prior tocontinued fluid flow through capillary channel 646 and into mixed fluidreceiving chamber 637. Mixed fluid receiving chamber 637 is from about0.02 mm to about 3 cm deep, has a cross-sectional diameter of from about0.02 mm to about 10 cm, and is positioned from about 1.2 cm to about 14cm from the center of rotation.

[0143] As illustrated in FIGS. 8A through 8D, in the use of thisplatform a volume of blood is applied to metering capillary 602, eitherdirectly or using the metering components of the platform describedabove. Blood flows into cell separation chamber 607 with displaced airflowing through air channel 654, and may be vented to the surface of thedisc or in communication with blood fluid chamber 604 (FIG. 8B). Asblood flows into cell separation chamber 607, the fluid component of theblood wicks into matrix 606; the pore size of the matrix (from about 0.2mm to about 2 μm) is chosen to prevent the cellular components of theblood from entering the matrix. In a preferred embodiment, matrix 606 isarranged in cell separation chamber 607 so that the matrix is in contactwith or more preferably adhered to the lower surface of cell separationchamber 607. Blood fluid, such as plasma or serum, traverses matrix 606by wicking and under rotation-induced pressure, saturating the matrixand filling a space formed between the top surface of the matrix and thetop surface of cell separation chamber 607 (FIG. 8C).

[0144] Blood fluid exits cell separation chamber 607 through capillary610 and flows into mixing chamber 615. Similarly, at a rotational speedof from about 200 rpm to about 200 rpm sufficient to overcome capillaryvalve 625, or upon release of sacrificial valve 625, solubilizedreagents 608 flow through capillary 620 and into mixing chamber 615(FIG. 8C). Fluid flow within mixing chamber 615 is turbulent, incontrast to fluid flow through capillaries 610 and 620, which isprimarily laminar, so that mixing occurs predominantly in mixing chamber615. Fluid flow proceeds through channel 636 and then either throughsecond mixing chamber 640 or directly through capillary 646 into mixedfluid receiving chamber 637 (FIG. 8D).

[0145] Glucose detection reagents mixed with blood in mixed fluidreceiving chamber 637 (FIG. 8D). The timescale over which thesereactions take place preferably goes to completion within about 0.5 min.to about 5 min. Reaction of the blood component(s) with reagents 608produce a colored product (FIG. 8D). Detection of the colored product ofthe glucose detecting reaction is performed in mixed fluid receivingchamber 637. In preferred embodiments, detection is performedspectrophotometrically, although visual inspection is also contemplatedin alternative embodiments of the invention. The amount of coloredproduct produced is detected and the amount of glucose in the bloodsample determined thereby.

[0146] The microfluidics structures of the invention are used to detectthe amount or concentration of a chemical species in a solution orcomplex mixture, most preferably an aqueous solution or mixture. Incertain preferred embodiments, the chemical species to be detected inglucose in blood. In one embodiment, blood glucose in blood is detectedusing a hexokinase assay.

[0147] In this assay, hexokinase converts glucose to glucose-6-phosphate(G-6-P) in the presence of magnesium cation. G-6-P is then converted to6-phosphogluconate (6-PG) by glucose-6-phosphate dehydrogenase in thepresence of NADP producing a stoichiometric amount of NADPH. NADPH isoxidized by reaction with phenazine methosulfate (PMS) as anintermediate, which is then oxidized by reaction with indotetrotazoliumchloride, which forms a colored product that absorbs visible light at520 nm. Optimum reagent component concentrations vary according to thespecifics of the application of this chemical assay, as well understoodand practiced by one versed in the art. This reaction scheme isillustrated as follows:

[0148] The amount of glucose in the blood fluid is directly related tothe amount of reduced indotetrotazolium chloride, and the concentrationof reduced indotetrotazolium chloride was related to glucoseconcentration using light spectroscopy as described below, as understoodby those with skill in the art. All of these reagents are preferablyprovided as dried reagents 108 applied to the disc, for example,comprising matrix 106 shown in FIGS. 1 through 8. These reagents can belyophilized or air dried directly onto the surface of the disk, forexample by “ink-jet” methods, or can be applied as dried beads or otherparticulate components.

[0149] In a variation of this reaction scheme, reduction of NADP isassayed directly without the use of PMS or indotetrazolium chloride. Inthis embodiment, the concentration of oxidized NADP is detectedspectrophotometrically by illuminating the sample with light at 340 nm,and detecting absorbance. The amount of glucose in the sample isinversely proportional to the amount of oxidized NADP present in thesample after reaction. In these embodiments, the amount of NADP must beprecisely controlled to be certain that the inverse proportionalitybetween oxidized NADP and glucose is maintained.

[0150] Detection is performed by transmission, reflection or reflectancespectroscopy. In transmission spectroscopy, light at a wavelength of 520nm produced by a narrow band light source, most preferably using a lightemitting diode (LED) with a filter, enters one face of an assay chamber,more preferably at a position in the chamber that comprises a detectioncell, and the light transmitted through the detection cell is detectedusing a photomultiplier tube, photodiode, photodiode array, or avalanchephotodiode. The photomultiplier tube is calibrated so that the amount oftransmitted light detected is interpreted according to Beer's law todetermine optical density and hence concentration of glucose in thesample by the well-known linear relation between the logarithm of theincident intensity/transmitted intensity and the concentration ofcolored (absorbing) product.

[0151] Alternatively, production of colored reaction products isdetected by reflection spectroscopy. Light at a wavelength of 520 nm isproduced by a narrow band light source, most preferably a furthercombination with a monochromator, grating, or filter. Monochromaticlight sources such as lasers or rare gas lamps as well asquasi-monochromatic light sources such as LEDs may also be used andenters one face of an assay chamber, more preferably at a position inthe chamber that comprises a detection cell. In preferred embodiments,the face of the assay chamber or detection cell opposite to the faceilluminated by the light source comprises a reflective surface,preferably formed using a reflective material, including but not limitedto an aluminum layer, a metallized glass, a mirror, and high glosspaint, or a diffusely reflecting surface such as TiO₂, underneath thecolored fluid, which advantageously decreases the contribution ofscratches or rotor wobble. Illuminated light is reflected back throughthe detection cell at a direct or through an oblique angle and isdetected using a photomultiplier tube photo diode, photodiode array, oravalanche photodiode.

[0152] For embodiments of the platforms of the invention wherein theassay chamber comprises a solid or porous matrix, production of coloredreaction products is most preferably detected by a variation onreflection spectroscopy described above. Light at a wavelength of 520 nmis produced by a narrow band light source, most preferably in furthercombination with a monochromator is produced that enters one face of anassay chamber, more preferably at a position in the chamber thatcomprises a detection cell. Light is absorbed from the colored reactionproducts comprising the matrix and is scattered from the matrix whichcomprises a diffusely-scattering material. The diffusely scattered,reflected light is detected.

[0153] In an alternative reaction protocol, glucose oxidase is used toproduce hydrogen peroxide by oxidation of glucose in the blood sample.In this reaction scheme, glucose oxidase converts glucose to gluconicacid and hydrogen peroxide; a twice-stoichiometric amount of hydrogenperoxide is produced relative to the amount of glucose present in theblood sample. The hydrogen peroxide then oxidizes a dye precursorpresent in the assay chamber or preferably within a detection cell,yielding a colored product. A variety of dye precursors are useful inthe practice of this aspect of the invention, including but not limitedto O-dianisidine, O-toluidine, O-tolidine, benzidine,2,2′-azinodi-(3-ethylbenzthiazoline sulfonic acid),3-methyl-2-benzthiazolinone hydrazone plus N,N-dimethylaniline, phenylplus 4-aminophenzanone, sulfonated 2,4-dichlorophenol plus4-aminophenzanone, 3-methyl-2-benzothiazolinone hydrazone plus3-(dimethylamino) benzoic acid, 2-methoxy-4-allyl phenol and4-aminoantipyrene-dimethylaniline. Optimum reagent componentconcentration vary according to the specifics of the application of thischemical assay, as well understood and practiced by one versed in theart. This reaction scheme is illustrated as follows:

[0154] The amount of glucose in the blood fluid is directly related tothe amount of oxidized dye produced. The concentration of oxidized dyeproduced is determined by Beers law and light spectroscopy measurements.The total amount of oxidized dye produced is then used to calculate thesample glucose concentration. Alternatively and preferably, the rate ofoxidized dye production is measured and that rate related to the sampleglucose concentration. Calibration, to relate glucose concentration tooptical measurements calculated by either method above, are wellunderstood by one versed in this art. All of these reagents arepreferably provided as dried reagents 108 applied to the disc, forexample, comprising matrix 106 shown in FIGS. 1 through 8. Thesereagents can be applied, by for example methods, including but notlimited to, filling, spraying, dipping, rolling and stamping a solutioncontaining reagent components followed by lyophilization or air drying.Several applications may be made sequentially, with differentcomponents.

[0155] The invention also provides microsystem platforms for performingseparations of particular components of a solution or complex mixture.In particular, the invention provides disc embodiments of the platformsof the invention comprising separation chambers containing components ormatrices that specifically bind and retain particular chemical speciescomprising a chemical solution or complex mixture. This aspect of theinvention is illustrated by a microfluidics array for separatingglycated hemoglobin from a blood sample, as shown in FIG. 9.

[0156] Construction of the disk embodiments of the platforms of theinvention were as described above. The blood application and meteringcomponents and their dimensions and relationships to one another areidentical to those described above, comprising sample entry port chamber701, metering capillary 702, overflow capillary 703, and overflowchamber 705. As in Example 1, each of the overflow and fluid chambers isalso connected with air ports or air channels, such as 754, andcapillary junction(s) 755, that permit venting of air displaced by fluidmovement on the platform.

[0157] Metering capillary 702 is fluidly connected to capillary 710 thatis from about 0.02 mm to about 2 mm deep and has a cross-sectionaldiameter of from about 0.02 mm to about 2 mm. Capillary 710 is furtherfluidly connected to mixing chamber 715 that is from about 0.02 mm toabout 3 cm deep, has a cross-sectional diameter of from about 0.02 mm toabout 10 cm, and is positioned from about 1.2 cm to about 14 cm from thecenter of rotation. Lysis buffer chamber 716 containing blood lysisbuffer is from about 0.02 mm to about 3 cm deep, has a cross-sectionaldiameter of from about 0.02 mm to about 10 cm, and is positioned fromabout 1.2 cm to about 14 cm from the center of rotation. Lysis bufferchamber 716 is positioned more proximally to the axis of rotation thatmixing chamber 715, and has a volumetric capacity of from about 15 μL toabout 150 μL of lysis buffer, composed of 0.1% Triton X100 in 50 mM TrispH 9.5. Lysis buffer chamber 715 is fluidly connected through capillary718 to mixing chamber 715.

[0158] Mixing chamber 715 is fluidly connected to capillary 717 that isfrom about 0.02 mm to about 2 mm deep and has a cross-sectional diameterof from about 0.02 mm to about 2 mm and is connected to secondarymetering structure 719. Secondary metering structure 719 is from about0.02 mm to about 3 cm deep, and is positioned from about 1.2 cm to about14 cm from the center of rotation. Secondary metering structure 719 isconstructed to comprise three sections. A first metering section isarranged proximal to the entry position of capillary 717 and isseparated from a second metering section by a septum that extends fromthe distal wall of the structure to a position just short of theproximal wall of the structure. This arrangement produces a fluidconnection between the first metering section having a volumetriccapacity of from about 5 μL to about 15 μL and second metering sectionhaving a volumetric capacity of from about 5 μL to about 15 μL. Anoverflow chamber is positioned adjacent to the second metering sectionand separated by a septum that extends from the distal wall of thestructure to a position just short of the proximal wall of thestructure. This arrangement produces a fluid connection between thesecond metering section and the overflow sections of the secondarymetering structure 719.

[0159] Capillary 721 is in fluid connection with secondary meteringstructure 719 at the distal wall of the first metering section.Capillary 721 is from about 0.02 mm to about 2 mm deep and has across-sectional diameter of from about 0.02 mm to about 2 mm and isfluidly connected to boronate affinity matrix chamber 722. Boronateaffinity matrix chamber 722 is from about 0.02 mm to about 0.3 cm deep,has a cross-sectional diameter of from about 0.02 mm to about 10 cm andis positioned from about 1.2 cm to about 14 cm from the axis ofrotation. Boronate affinity matrix chamber 722 further comprises 20-50μL boronate-functionalized agarose beads having a mean diameter of about60 μm; the beads are maintained in the chamber 722 using a porous frit727. Fluid flow through capillary 721 is connected to capillary orsacrificial valve 723. Capillary 724 is in fluid connection withsecondary metering structure 719 at the distal wall of the secondmetering section. Capillary 724 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02 mm to about 2 mmand is connected to read window 726. Read window 726 is from about 0.02mm to about 3 cm deep, has a cross-sectional diameter of from about 0.02mm to about 10 cm and is positioned from about 1.2 cm to about 14 cmfrom the axis of rotation. Read window is comprised of a materialtransparent to light at a wavelength of about 430 nm. Fluid flow throughcapillary 724 is connected to capillary or sacrificial valve 725.

[0160] Boronate affinity matrix chamber 722 is further fluidly connectedto capillary 728. Capillary 728 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02 mm to about 2 mmand is connected to column preparation buffer reservoir 729. Columnpreparation buffer reservoir 729 is from about 0.02 mm to about 3 cmdeep and has a cross-sectional diameter of from about 0.02 mm to about10 cm and is positioned from about 1.2 cm to about 12 cm from the axisof rotation, more proximal than boronate affinity matrix chamber 722.Column preparation buffer reservoir 729 comprises from about 100 μL toabout 500 μL of column preparation buffer comprising magnesium chloride,taurine, D,L-methionine, sodium hydroxide, antibiotics and stabilizers(obtained from IsoLab as described in the Examples below). Fluid flowthrough capillary 728 is connected to capillary or sacrificial valve735.

[0161] Boronate affinity matrix chamber 722 is further fluidly connectedto capillary 730. Capillary 730 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02mm to about 2 mmand is connected to column wash buffer reservoir 731. Column wash bufferreservoir 731 is from about 0.02 mm to about 3 cm deep and has across-sectional diameter of from about 0.02 mm to about 10 cm and ispositioned from about 1.2 cm to about 14 cm from the axis of rotation,more proximal than boronate affinity matrix chamber 722. Column washbuffer reservoir 731 comprises from about 100 μL to about 500 μL ofcolumn preparation buffer as described above. Fluid flow throughcapillary 730 is connected to capillary or sacrificial valve 736.

[0162] In alternative embodiments, column preparation buffer reservoir729 and column wash buffer reservoir 731 can be the same reservoir, orcan be fluidly connected as shown in FIG. 9A.

[0163] Boronate affinity matrix chamber 722 is further fluidly connectedto capillary 732. Capillary 732 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02 mm to about 2 mmand is connected to read window 726.

[0164] Read window 726 is further fluidly connected to capillary 733.Capillary 733 is from about 0.02 mm to about 2 mm deep and has across-sectional diameter of from about 0.02 mm to about 2 mm and isconnected to waste reservoir 734. Waste reservoir 734 is from about 0.02mm to about 3 cm deep and has a cross-sectional diameter of from about0.02 mm to about 10 cm and is positioned from about 1.2 cm to about 14cm from the axis of rotation.

[0165] As illustrated in FIG. 9A, in the use of this platform a volumeof blood from about 15 μL to about 150 μL is applied to meteringcapillary 702, either directly or using the metering components of theplatform described above. Blood flowing through capillary 710 and lysisbuffer flowing through capillary 718 are mixed in mixing chamber 715 byovercoming capillary valve 711 or release of sacrificial valve 711. Avolume of lysis buffer from about 25 μL to about 90 μL was mixed withthe blood sample. Fluid-flow within mixing chamber 715 is turbulent, incontrast to fluid flow through capillaries 710 and 718, which isprimarily laminar, so that mixing occurs predominantly in mixing chamber715. Fluid flow proceeds through channel 717 and into secondary meteringstructure 719.

[0166] The mixture of lysis buffer and blood, comprising a lysed bloodsample, flows at a rotational speed f₂ from about 200 rpm to about 2000rpm into secondary metering structure 719. The lysed blood sample entersand fills the first section of secondary metering structure 719.Continued lysed blood sample flow into secondary metering structure 719then fills the second section of secondary metering structure 719. Anyadditional lysed blood sample then empties into the overflow chamber ofsecondary metering structure 719. Most preferably, a sufficient volumeof lysis buffer and blood sample is applied to the disc to fill at leastthe first and second metered sections of secondary metering structure719.

[0167] After the lysed blood sample is completely transferred tosecondary metering structure 719, capillary or sacrificial valve 735 isreleased, allowing from 100 μL to about 500 μL of column preparationbuffer to flow at rotational speed f₂ through capillary 730 and intoboronate affinity matrix 722. Continued or discontinuous rotationmotivates column preparation buffer through boronate affinity matrix722, capillary 732, read window 726, capillary 733 and into wastereservoir 734.

[0168] After the column preparation buffer is applied to boronateaffinity matrix chamber 722, capillary or sacrificial valve 723 isreleased, allowing the metered lysed blood sample from the first meteredsection of secondary metering structure 719 through capillary 721 andinto boronate affinity matrix 722 and allowed to incubate in theaffinity matrix chamber for from about 0.5 to about 5 min. Capillary orsacrificial valve 736 is then released, allowing from 100 μL to about500 μL of column wash buffer to flow at rotational speed f_(?) throughcapillary 730 and into boronate affinity matrix 722. Continued ordiscontinuous rotation motivates column preparation buffer throughboronate affinity matrix 722, capillary 732 and into read window 726.During fluid flow of wash buffer through the boronate affinity matrixchamber, read window is preferably illuminated by light at a wavelengthof 430 nm and the concentration of hemoglobin in the sample afterglycated hemoglobin has been removed by the boronate affinity matrix isdetermined thereby.

[0169] Capillary or sacrificial valve 725 is released at rotationalspeed f₃ of from about 100-1000 rpm and the metered lysed blood samplefrom the second metered section of secondary metering structure 719flows through capillary 724 and into read window 726. Read window isthen illuminated by light at a wavelength of 430 nm and theconcentration of hemoglobin in the sample determined transmission,reflection, or reflectance spectroscopy. The amount of glycatedhemoglobin in the sample is determined by subtracting the amount ofhemoglobin obtained in the first reading from the amount of hemoglobinobtained in the second reading.

[0170] An alternative embodiment of the glycated hemoglobin assaymicrosystem platform of the invention is shown in FIG. 10. Constructionof the disk embodiments of the platforms of the invention were asdescribed above. The blood application and metering components and theirdimensions and relationships to one another are identical to thosedescribed above, comprising sample entry port chamber 801, meteringchamber 802, overflow capillary 803, and overflow chamber 805. As inExample 1, each of the overflow and fluid chambers is also connectedwith air ports or air channels, such as 854, and capillary junction(s)855, that permit venting of air displaced by fluid movement on theplatform.

[0171] Metering capillary 802 is fluidly connected to capillary 810 thatis from about 0.02 mm to about 2 mm deep and has a cross-sectionaldiameter of from about 0.02 mm to about 2 mm. Capillary 810 is furtherfluidly connected to mixing chamber 815 that is from about 0.02 mm toabout 3 cm deep, has a cross-sectional diameter of from about 0.02 mm toabout 10 cm, and is positioned from about 1.2 cm to about 14 cm from thecenter of rotation. Lysis buffer chamber 816 containing blood lysisbuffer is from about 0.02 mm to about 3 cm deep, has a cross-sectionaldiameter of from about 0.02 mm to about 10 cm, and is positioned fromabout 1.2 cm to about 14 cm from the center of rotation. Lysis bufferchamber 816 is positioned more proximally to the axis of rotation thatmixing chamber 815, and has a volumetric capacity of from about 15 μL toabout 150 μL of lysis buffer, composed of 0.1% Triton X100 in 50 mM TrispH 9.5. Lysis buffer chamber 815 is fluidly connected through capillary818 to mixing chamber 815.

[0172] Mixing chamber 815 is fluidly connected to capillary 817 that isfrom about 0.02 mm to about 2 mm deep and has a cross-sectional diameterof from about 0.02 mm to about 2 mm and is connected to secondarymetering structure 819. Secondary metering structure 819 is from about0.02 mm to about 3 cm deep, and is positioned from about 1.2 cm to about14 cm from the center of rotation. Secondary metering structure 819 isconstructed to comprise two sections. A metering section is arrangedproximal to the entry position of capillary 817 and is separated from anoverflow section by a septum that extends from the distal wall of thestructure to a position just short of the proximal wall of thestructure. This arrangement produces a fluid connection between themetering section having a volumetric capacity of from about 5 μL toabout 15 μL and the overflow chamber.

[0173] Capillary 821 is in fluid connection with secondary meteringstructure 819 at the distal wall of the first metering section.Capillary 821 is from about 0.02 mm to about 2 mm deep and has across-sectional diameter of from about 0.02 mm to about 2 mm and isfluidly connected to boronate affinity matrix chamber 822. Boronateaffinity matrix chamber 822 is from about 0.02 mm to about 0.3 cm deep,has a cross-sectional diameter of from about 0.02 mm to about 10 cm andis positioned from about 1.2 cm to about 14 cm from the axis ofrotation. Boronate affinity matrix chamber 822 further comprises 20-50μL boronate-functionalized agarose beads having a mean diameter of about60 μm; the beads are maintained in the chamber 822 using a porous frit827. Boronate affinity matrix chamber 822 further comprises at least onesurface that is translucent to light of at least wavelength of about 430nm, permitting direct illumination and interrogation of the amount ofglycated hemoglobin that is bound thereto. Fluid flow through capillary821 is connected to capillary or sacrificial valve 23.

[0174] Boronate affinity matrix chamber 822 is further fluidly connectedto capillary 828. Capillary 828 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02 mm to about 2 mmand is connected to column preparation buffer reservoir 829. Columnpreparation buffer reservoir 829 is from about 0.02 mm to about 3 cmdeep and has a cross-sectional diameter of from about 0.02 mm to about10 cm and is positioned from about 1.2 cm to about 12 cm from the axisof rotation, more proximal than boronate affinity matrix chamber 822.Column preparation buffer reservoir 829 comprises from about 100 μL toabout 500 μL of column preparation buffer comprising magnesium chloride,taurine, D,L-methionine, sodium hydroxide, antibiotics and stabilizers(obtained from IsoLab as described in the Examples below). Fluid flowthrough capillary 828 is connected to capillary or sacrificial valve835.

[0175] Boronate affinity matrix chamber 822 is further fluidly connectedto capillary 830. Capillary 830 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02 mm to about 2 mmand is connected to column wash buffer reservoir 831. Column wash bufferreservoir 831 is from about 0.02 mm to about 3 cm deep and has across-sectional diameter of from about 0.02 mm to about 10 cm and ispositioned from about 1.2 cm to about 14 cm from the axis of rotation,more proximal than boronate affinity matrix chamber 822. Column washbuffer reservoir 831 comprises from about 100 μL to about 500 μL ofcolumn preparation buffer as described above. Fluid flow throughcapillary 830 is connected to capillary or sacrificial valve 836.

[0176] In alternative embodiments, column preparation buffer reservoir829 and column wash buffer reservoir 831 can be the same reservoir, orcan be fluidly connected as shown in FIG. 10A.

[0177] Boronate affinity matrix chamber 822 is further fluidly connectedto capillary 832. Capillary 732 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02 mm to about 2 mm.Capillary 832 is further fluidly connected with waste reservoir 834.Waste reservoir 834 is from about 0.02 mm to about 3 cm deep and has across-sectional diameter of from about 0.02 mm to about 10 cm and ispositioned from about 1.2 cm to about 14 cm from the axis of rotation.

[0178] As illustrated in FIG. 10A, in the use of this platform a volumeof blood from about 15 μL to about 150 μL is applied to meteringcapillary 802, either directly or using the metering components of theplatform described above. Blood flowing through capillary 810 and lysisbuffer flowing through capillary 818 are mixed in mixing chamber 815 byovercoming capillary valve 811 or release of sacrificial valve 811. Avolume of lysis buffer from about 25 μL to about 90 μL was mixed withthe blood sample. Fluid flow within mixing chamber 815 is turbulent, incontrast to fluid flow through capillaries 810 and 818, which isprimarily laminar, so that mixing occurs predominantly in mixing chamber815. Fluid flow proceeds through channel 817 and into secondary meteringstructure 819.

[0179] The mixture of lysis buffer and blood, comprising a lysed bloodsample, flows at a rotational speed f₂ from about 200 rpm to about 2000rpm into secondary metering structure 819. The lysed blood sample entersand fills the metering section of secondary metering structure 819. Anyadditional lysed blood sample then empties into the overflow chamber ofsecondary metering structure 819. Most preferably, a sufficient volumeof lysis buffer and blood sample is applied to the disc to fill at leastthe metered sections of secondary metering structure 819.

[0180] After the lysed blood sample is completely transferred tosecondary metering structure 819, capillary or sacrificial valve 835 isreleased, allowing from 100 μL to about 500 μL of column preparationbuffer to flow at rotational speed f₂ through capillary 830 and intoboronate affinity matrix 822. Continued or discontinuous rotationmotivates column preparation buffer through boronate affinity matrix822, capillary 832, and into waste reservoir 834.

[0181] After the column preparation buffer is applied to boronateaffinity matrix chamber 822, capillary or sacrificial valve 823 isreleased, allowing the metered lysed blood sample from the first meteredsection of secondary metering structure 819 through capillary 821 andinto boronate affinity matrix 822 and allowed to incubate in theaffinity matrix chamber for from about 0.5 to about 5 min. Capillary orsacrificial valve 836 is then released, allowing from 100 μL to about500 μL of column wash buffer to flow at rotational speed f_(?) throughcapillary 830 and into boronate affinity matrix 822. Continued ordiscontinuous rotation motivates column preparation buffer throughboronate affinity matrix 822, capillary 732 and into waste reservoir834. During fluid flow of wash buffer through the boronate affinitymatrix chamber, the chamber is preferably illuminated by light at awavelength of 430 nm through the translucent portion of the chamber. Theconcentration of glycated hemoglobin in the sample is determinedthereby.

[0182] An alternative embodiment of the glycated hemoglobin assaymicrosystem platform of the invention is shown in FIG. 11. Constructionof the disk embodiments of the platforms of the invention were asdescribed above. The blood application and metering components and theirdimensions and relationships to one another are identical to thosedescribed above, comprising sample entry port chamber 901, meteringcapillary 902, overflow capillary 903, and overflow chamber 905. As inExample 1, each of the overflow and fluid chambers is also connectedwith air ports or air channels, such as 954, and capillary junction(s)955, that permit venting of air displaced by fluid movement on theplatform.

[0183] Metered capillary 902 is fluidly connected to capillary 910 thatis from about 0.02 mm to about 2 mm deep and has a cross-sectionaldiameter of from about 0.02 mm to about 2 mm and is connected tosacrificial wax valve 911. Sacrificial wax valve 911 is further fluidlyconnected with capillary 912 that is from about 0.03 mm to about 2.2 mm.Capillary 912 is further fluidly connected to blood lysis chamber 915that is from about 0.02 mm to about 3 cm deep, has a cross-sectionaldiameter of from about 0.02 mm to about 10 cm, is positioned from about1.2 cm to about 14 cm from the center of rotation, and contains fromabout 25 μL to about 90 μL of blood lysis solution (0.1% Triton-X100 in50 mM Tris, pH 9.5). Blood lysis chamber 915 is further fluidlyconnected with capillary 918 that is from about 0.02 mm to about 2 mmdeep and has a cross-sectional diameter of from about 0.02 mm to about 2mm and is connected to sacrificial wax valve 913. Sacrificial wax valve913 is further fluidly connected with wax recrystallization chamber 914that is from about 0.02 mm to about 2 mm deep and has a volumetriccapacity sufficient to sequester melted wax from a released wax valveand prevent occlusion of the lumen of the capillary 918 controlled bythe valve.

[0184] Capillary 918 is further fluidly connected to secondary meteringstructure 919. Secondary metering structure 919 is from about 0.02 mm toabout 3 cm deep, and is positioned from about 1.2 cm to about 14 cm fromthe center of rotation. Secondary metering structure 919 is constructedto comprise three sections. A first section comprises a throwawaysection sample having a volumetric capacity of from about 5 μL to about10 μL because it is thought that by taking the second section a morerepresentative would be obtained. This throwaway section is arrangedproximal to the entry position of capillary 918 and is separated from ametering section by a septum that extends from the distal wall of thestructure to a position just short of the proximal wall of thestructure. This arrangement produces a fluid connection between thethrowaway section and the metering section. The metering section has avolumetric capacity of from about 5 μL to about 10 μL and is fluidlyconnected to an overflow section having an excess volumetric capacity offrom about 15 μL to about 150 μL. The volumetric capacity of theoverflow section is sufficient to accommodate the largest blood fluidvolume applied to the disk.

[0185] Capillary 921 is in fluid connection with secondary meteringstructure 919 at the distal wall of the metering section. Capillary 921is from about 0.02 mm to about 2 mm deep and has a cross-sectionaldiameter of from about 0.02 mm to about 2 mm and is connected toboronate affinity matrix chamber 922. Boronate affinity matrix chamber922 is from about 0.02 mm to about 3 cm deep, has a cross-sectionaldiameter of from about 0.02 mm to about 10 cm and is positioned fromabout 1.2 cm to about 14 cm from the axis of rotation. Boronate affinitymatrix chamber 922 further comprises 10-50 μL of boronate-functionalizedagarose beads having a mean diameter of about 60 cm; the beads aremaintained in the chamber 922 using a porous frit 927. Fluid flowthrough capillary 921 is connected to sacrificial valve 923. Sacrificialwax valve 923 is further fluidly connected with wax recrystallizationchamber 924 that is from about 0.03 mm to about 2.2 mm deep and has avolumetric capacity sufficient to sequester melted wax from a releasedwax valve and prevent occlusion of the lumen of the capillary 923controlled by the valve.

[0186] Boronate affinity matrix chamber 922 is further fluidly connectedto capillary 928. Capillary 928 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02 mm to about 2 mmand is connected to column wash buffer reservoir 929. Column wash bufferreservoir 929 is from about 0.02 mm to about 3 cm deep and has across-sectional diameter of from about 0.02 mm to about 10 cm and ispositioned from about 1.2 cm to about 14 cm from the axis of rotation,more proximal than boronate affinity matrix chamber 922. Column washbuffer reservoir 929 comprises from about 100 μL to about 500 μL ofcolumn wash buffer as described above. Fluid flow through capillary 928is connected to sacrificial valve 936. Sacrificial wax valve 936 isfurther fluidly connected with wax recrystallization chamber 937 that isfrom about 0.03 mm to about 2.2 mm deep and has a volumetric capacitysufficient to sequester melted wax from a released wax valve and preventocclusion of the lumen of the capillary 928 controlled by the valve.

[0187] Boronate affinity matrix chamber 922 is further fluidly connectedto capillary 932. Capillary 932 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02 mm to about 10 cmand is connected to sample collection cuvette array 934. Samplecollection cuvette array 934 is from about 0.02 mm to about 3 cm deepand has a cross-sectional diameter of from about 0.02 mm to about 10 cmand is positioned from about 1.2 cm to about 14 cm from the axis ofrotation. Sample collection cuvette array 934 is separated into amultiplicity of individual chambers, each separated from one another bysepta that extend from the distal wall of the cuvettes to a positionadjacent to the proximal wall of the cuvettes, so that a fluid passage950 is maintained between each of the cuvettes. The fluid passage 950 isformed by the back (proximal wall) of the sample collection cuvettearray 934 and the row of septa separating each of the sections of thesample collection cuvettes 934. Capillary 932 is fluidly connected tosample collection cuvette array 934 at a position adjacent to theproximal wall of the array and directed to the cuvette most proximal tothe boronate affinity matrix chamber 922. Alternatively, the septa canbe eliminated in sample collection cuvette array 934, wherein the samplecuvette is a single chamber.

[0188] Capillary 941 is fluidly connected to secondary meteringstructure 919. Capillary 941 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02 mm to about 2 mmand is connected to secondary metering structure 919 at a positionbetween the metering section and the overflow section. Capillary 941 isfurther fluidly connected with total hemoglobin read chamber 942. Totalhemoglobin read chamber 942 is from about 0.02 mm to about 3 cm deep, ispositioned from about 1.2 cm to about 14 cm from the center of rotation,and has a volumetric capacity of from about 50 μL to about 250 μL. Totalhemoglobin read chamber 942 is positioned radially more distal from thecenter of rotation than secondary metering structure 919, and comprisesa read window translucent to light having a wavelength of from about 430nm. In addition, there is no capillary or sacrificial valvingcontrolling fluid flow in capillary 941.

[0189] The platform also comprises control sample read cuvettes 943 and944, advantageously positioned in proximity to total hemoglobin readchamber 942. Control sample read cuvettes 943 and 944 are each fromabout 0.02 mm to about 3 cm deep, positioned from about 1.2 cm to about14 cm from the center of rotation, and have a volumetric capacity offrom about 50 μL to about 200 μL. Control sample read cuvettes 943 and944 comprise a read window translucent to light having a wavelength offrom about 430 nm. Control sample read cuvettes 943 and 944 are notfluidly connected to any other structure on the platform and containstandards and/or calibration reagents.

[0190] As illustrated in FIG. 11, in the use of this platform a volumeof blood from about 15 μL to about 150 μL is applied to meteringcapillary 902, either directly or using the metering components of theplatform described above. Release of sacrificial valve 911 and rotationof the platform at a rotational speed f₁ of from about 50 rpm to about1000 rpm motivates blood flow through capillary 910 and into blood lysischamber 915. The mixture of blood and blood lysis buffer in blood lysischamber 915 is mixed by agitation, wherein the platform is acceleratedrepeatedly from about +2000 rpm/sec to −2000 rpm/sec (wherein “+” and“−” indicate rotation in different directions) over a time period ofabout 30 sec to about 600 sec. Release of sacrificial valve 913 androtation of the platform at a rotational speed f₂ of from about 200 rpmto about 2000 rpm motivates the lysed blood sample to flow throughcapillary 918 and into secondary metering structure 919. Continuedrotation motivates lysed blood solution to fill the metering section ofsecondary metering structure 919; after filling of this section of thestructure, excess lysed blood sample flows through capillary 941 andinto total hemoglobin read chamber 942. After filling of totalhemoglobin read chamber 942, any excess lysed blood sample is displacedinto the overflow section of secondary metering structure 919.

[0191] Release of sacrificial valve 923 and rotation of the platform ata rotational speed f₃ of from about 200 rpm to about 2000 rpm motivatesthe metered lysed blood sample in the metering section of secondarymetering structure 922 to flow through capillary 921 and into boronateaffinity matrix chamber 922. After incubation of the metered lysed bloodsample 956 in boronate affinity matrix chamber 922, sacrificial valve936 is released and the platform is rotated at a rotational speed f₄ offrom about 200 rpm to about 3000 rpm. Column wash buffer flows fromcolumn wash buffer reservoir 929 through capillary 921 and into boronateaffinity matrix chamber 922, displacing the unbound hemoglobin fractionthrough capillary 932 and into sample collection cuvette array 934.Continued rotation of the platform displaces the collected samplesequentially into the separate cuvettes radially away from the positionof boronate affinity matrix chamber 922 on the platform. Samplecollection cuvette array 934 is then interrogated by illumination withlight at a wavelength of 430 nm, and the concentration of non-glycatedhemoglobin in the blood sample determined. Additionally, illumination oftotal hemoglobin read chamber 942 with light at a wavelength at 415 nmis performed to determine the concentration of total hemoglobin in theblood sample. The amount of glycated hemoglobin is calculated bysubtracting the amount of non-glycated hemoglobin in the sample from thetotal hemoglobin concentration in the sample. Control sample readcuvettes 943 and 944 are used to calibrate the spectrophotometricreadings.

[0192] In alternative embodiments of these microfluidics systems, theboronate affinity matrix is replaced by other substances capable ofdifferentially binding glycated or non-glycated hemoglobin species. In afirst embodiment of such an alternative, m-aminophenylboronatepolyacrylic acid is used to derivatize a positively-charged nylon 66membrane (such as Biodyne-B, Pall Biosupport Division, Port Washington,N.Y.). This membrane is used in substitution for theboronate-functionalized agarose beads in the boronate affinity matrixchambers of the invention. In preferred embodiments, the boronateaffinity matrix chambers are modified to contain the membrane in contactwith or more preferably adhered to the platform surface within thechamber, so that one face of the membrane derivatized withm-aminophenylboronate polyacrylic acid is in contact with the lysedblood sample. Binding of glycated hemoglobin to the membrane isquantitated by visible light reflectance spectroscopy at a wavelength of415 nm.

[0193] A second alternative embodiment of the glycated hemoglobinmicrosystems assays of the invention comprises inositol hexaphosphate.In these embodiments, inositol hexaphosphate is attached (covalently orby electrostatic interactions) to a solid support, including but notlimited to beads, membranes, pads, etc. The lysed blood sample istreated with sodium dithionite to convert it to the deoxy form. In theMicrosystems platforms of the invention, an effective amount of sodiumdithionite is provided with the lysis buffer, or as a component of thesecondary metering structures, most preferably as a dry powder coatingon the walls of one or both of the metering sections thereof. Thedeoxygenated lysed blood sample is then placed in contact with the solidsupport comprising inositol hexaphosphate, preferably comprising and insubstitution for the boronate affinity matrix chamber of the glycatedhemoglobin platforms of the invention. In these embodiments, the portionof hemoglobin that does not bind to the inositol phosphate-containingsolid support is the glycated fraction, which can be delivered to areads chamber, cuvette or other optically-appropriate component of theplatform and the amount of glycated hemoglobin determine directly byvisible light reflectance spectrophotometry at a wavelength of 415 nm.

[0194] The invention also provides microsystem platforms for performinga multiplicity of reactions including identification of chemical speciesfrom solutions or complex mixtures and separations of particularcomponents of a solution or complex mixture. This aspect of theinvention is illustrated by a microfluidics array for determiningglucose concentration and separating glycated hemoglobin from a bloodsample, as shown in FIGS. 12A through 12Q and 13A through 13D.

[0195] Construction of the disk embodiments of the platforms of theinvention were as described above. FIG. 13B shows a detailed descriptionof the microfluidics components of the platform, which are described inadditional detail below. FIG. 13C shows the geometry of a screen printedelectrical lead layer deposited on a mylar substrate. FIG. 13D shows thepositions of screen printed heaters activated by the electrical leads ofthe lead layer and screen printed on mylar. FIG. 13E shows a overlay ofthese components in the assembled disc.

[0196] Referring to the microfluidics components of the platform shownin FIG. 13B, an entry port 1 is positioned on the top surface of thedisc and is open for the user to apply an unmetered sample. Entry port 1is from about 0.02 mm to about 3 cm deep, has a cross-sectional diameterof from about 0.02 mm to about 10 cm, is positioned from about 1.2 cm toabout 14 cm from the center of rotation, and is fluidly connected tocapillary channel 1A that is from about. 0.02 mm to about 2 mm deep andhas a cross-sectional diameter of from about 0.02 mm to about 2 mm.Capillary channel 1A is fluidly connected to metering component 2, whichcomprises four sections. The first section is rectangularly-shaped andextends in a direction proximal to the axis of rotation away from itsfluid connection with capillary channel 1A. About half way up thisrectangular section is a lateral chamber 3, which empties into a bloodglucose metering chamber 4 and an overflow chamber 5. The first sectionof the metering component is from about 0.02 mm to about 3 cm deep, hasa cross-sectional diameter of from about 0.02 mm to about 10 cm, ispositioned from about 1.2 cm to about 14 cm from the center of rotation,and has a volumetric capacity of from about 15 μL to about 150 μL. Thelateral chamber 3 is from about 0.02 mm to about 3 cm deep, has across-sectional diameter of from about 0.02 mm to about 10 cm, and ispositioned from about 1.2 cm to about 14 cm from the center of rotation.Blood glucose metering chamber 4 is from about 0.02 mm to about 3 cmdeep, has a cross-sectional diameter of from about 0.02 mm to about 10cm, is positioned from about 1.2 cm to about 14 cm from the center ofrotation and has a volumetric capacity of from about 1 μL to about 15μL. Overflow chamber 5 is from about 0.02 mm to about 3 cm deep, has across-sectional diameter of from about 0.02 mm to about 10 cm, ispositioned from about 1.2 cm to about 14 cm from the center of rotationand has a volumetric capacity of from about 5 μL to about 50 μL.

[0197] Overflow chamber 5 is fluidly connected to overflow channel 8that is from about 0.02 mm to about 3 cm deep, has a cross-sectionaldiameter of from about 0.02 mm to about 10 cm and extends from about 10mm to about 5 cm from overflow chamber 5. Overflow channel 5 is fluidlyconnected to short sample detection cuvette 9 that is from about 0.02 mmto about 3 cm deep, has a cross-sectional diameter of from about 0.02 mmto about 10 cm, is positioned from about 1.2 cm to about 10 cm from thecenter of rotation and has a volumetric capacity of from about 15 μL toabout 150 μL Blood glucose metering chamber 4 is fluidly connected tocapillary 15 that is from about 0.02 mm to about 3 cm deep, has across-sectional diameter of from about 0.02 mm to about 10 cm andextends from about 1 mm to about 5 cm from blood glucose meteringchamber 4. Capillary 15 is connected to sacrificial wax valve 6, whichis further fluidly connected with wax recrystallization chamber 6A thatis from about 0.03 mm to about 2.2 mm deep and has a volumetric capacitysufficient to sequester melted wax from a released wax valve and preventocclusion of the lumen of the capillary 15 controlled by the valve.Capillary 15 is further fluidly connected with glucose assay chamber 11that is from about 0.02 mm to about 3 cm deep, has a cross-sectionaldiameter of from about 0.02 mm to about 10 cm, is positioned from about1.2 cm to about 14 cm from the center of rotation and has a volumetriccapacity of from about 5 μL to about 50 μL. Glucose assay chamber 11comprises a depression 11A in the surface of the platform having a depthof from about 0.02 mm to about 3 cm, most preferably comprising acircular or concave depression connected to capillary 15 so that bloodflows into the chamber through the bottom of depression 11A. Depression11A is constructed to have a volumetric capacity of from half to twicethe assay volume. Blood glucose assay chamber 11 also comprises a pad ormatrix 10 of a hydrophilic substance possessing a pore size of 0.2-2.0μm, most preferably comprising a positively-charged nylon matrix havinga pore size of about 0.8 μm. The upper limit on pore size of matrix 10is chosen to inhibit or prevent blood cell entry into the matrix. Thematrix is positioned in blood glucose assay chamber 11 to be in fluidiccontact with depression 11A, more preferably covering depression 11A,and most preferably having a surface area greater than the surface areaof depression 11A. The matrix was further impregnated with immobilizedreagents 11B which produce a detectable product proportional to theamount or concentration of glucose in a blood sample. Most preferably,the detectable product is a colored product 11C, i.e., a productabsorbing light at a detectable, most preferably a visible, wavelength.

[0198] Lysis metering chamber 2 is fluidly connected to capillary 7controlled by sacrificial valve 7A. Sacrificial wax valve 7A is furtherfluidly connected with wax recrystallization chamber 7B that is fromabout 0.03 mm to about 2.2 mm deep and has a volumetric capacitysufficient to sequester melted wax from a released wax valve and preventocclusion of the lumen of the capillary 7 controlled by the valve.Capillary 7 is from about 0.02 mm to about 2 mm deep, has across-sectional diameter of from about 0.02 mm to about 2 mm, extendsfrom about 1 mm to about 5 cm from lysis metering chamber 2 and isfluidly connected to blood lysis chamber 16. Blood lysis chamber 16 isfrom about 0.02 mm to about 3 cm deep, has a cross-sectional diameter offrom about 0.02 mm to about 10 cm, is positioned from about 1.2 cm toabout 14 cm from the center of rotation, and contains from about 25 μLto about 90 μL of blood lysis solution (0.1% Triton-X100 in 50 mM Tris,pH 9.5).

[0199] Blood lysis chamber 16 is fluidly connected at a distal aspect tocapillary 17 controlled by sacrificial valve 18. Capillary 17 is fromabout 0.02 mm to about 2 mm deep and has a cross-sectional diameter offrom about 0.02 mm to about 2 mm Sacrificial wax valve 18 is furtherfluidly connected with wax recrystallization chamber 18A that is fromabout 0.03 mm to about 2.2 mm deep and has a volumetric capacitysufficient to sequester melted wax from a released wax valve and preventocclusion of the lumen of the capillary 17 controlled by the valve.Capillary 17 is fluidly connected to secondary metering structure 19.Secondary metering structure 19 is from about 0.02 mm to about 3 cmdeep, and is positioned from about 1.2 cm to about 14 cm from the centerof rotation. Secondary metering structure 19 is constructed to comprisethree sections. A first section 20 comprises a throwaway section havinga volumetric capacity of from about 5 μL to about 10 μL because it isthought that a more representative sample would be obtained thereby.Throwaway section 20 is arranged proximal to the entry position ofcapillary 17 and is separated from a metering section 21 by a septumthat extends from the distal wall of the structure to a position justshort of the proximal wall of the structure. This arrangement produces afluid connection between throwaway section 20 and the metering section21. Metering section has a volumetric capacity of from about 5 μL toabout 10 μL and is fluidly connected to an overflow section 24 having anexcess volumetric capacity of from about 15 μL to about 150 μL. Thevolumetric capacity of the overflow section is sufficient to accommodatethe largest blood fluid volume applied to the disk.

[0200] Capillary 25 is in fluid connection with secondary meteringstructure 21 at the distal wall of the metering section. Capillary 25 isfrom about 0.02 mm to about 2 mm deep and has a cross-sectional diameterof from about 0.02 mm to about 2 mm and is connected to boronateaffinity matrix chamber 28. Capillary 25 is fluidly connected tosacrificial wax valve 26 that is further fluidly connected with waxrecrystallization chamber 26A Wax recrystallization chamber 26A is fromabout 0.03 mm to about 2.2 mm deep and has a volumetric capacitysufficient to sequester melted wax from a released wax valve and preventocclusion of the lumen of the capillary 25 controlled by the valve.

[0201] Boronate affinity matrix chamber 28 is from about 0.02 mm toabout 3 cm deep, has a cross-sectional diameter of from about 0.02 mm toabout 10 cm and is positioned from about 1.2 cm to about 14 cm from theaxis of rotation. Boronate affinity matrix chamber 28 further comprisesboronate-functionalized agarose beads having a mean diameter of about 60μm; the beads are maintained in the chamber 28 using a porous frit 29.Boronate affinity matrix chamber 28 is further fluidly connected tocapillary 31. Capillary 31 is from about 0.02 mm to about 2 mm deep andhas a cross-sectional diameter of from about 0.02 mm to about 2 mm andis connected to column wash buffer reservoir 30. Column wash bufferreservoir 30 is from about 0.02 mm to about 3 cm deep and has across-sectional diameter of from about 0.02 mm to about 10 cm and ispositioned from about 1.2 cm to about 14 cm from the axis of rotation,more proximal than boronate affinity matrix chamber 28. Column washbuffer reservoir 30 comprises from about 250 μL to about 350 μL ofcolumn wash buffer as described above. Fluid flow through capillary 31is connected to sacrificial valve 32. Sacrificial wax valve 32 isfurther fluidly connected with wax recrystallization chamber 32A that isfrom about 0.03 mm to about 2.2 mm deep and has a volumetric capacitysufficient to sequester melted wax from a released wax valve and preventocclusion of the lumen of the capillary 32 controlled by the valve.

[0202] Boronate affinity matrix chamber 28 is further fluidly connectedto capillary 37. Capillary 37 is from about 0.02 mm to about 2 mm deepand has a cross-sectional diameter of from about 0.02 mm to about 2 mmand is connected to sample collection cuvette array 12. Samplecollection cuvette array 12 is from about 0.02 mm to about 3 cm deep andhas a cross-sectional diameter of from about 0.02 mm to about 10 cm andis positioned from about 1.2 cm to about 14 cm from the axis ofrotation. Sample collection cuvette array 12 is separated into amultiplicity of individual chambers, each separated from one another bysepta that extend from the distal wall of the cuvettes to a positionadjacent to the proximal wall of the cuvettes, so that a fluid passage50 is maintained between each of the cuvettes. The fluid passage 50 isformed by the back (proximal wall) of the sample collection cuvettearray 12 and the row of septa separating each of the sections of thesample collection cuvettes 12. Capillary 33 is fluidly connected tosample collection cuvette array 12 at a position adjacent to theproximal wall of the array and directed to the cuvette most proximal tothe boronate affinity matrix chamber 28. In alternative embodiments,collection cuvette array 12 can be constructed without such septa, andthis structure is then just a single collection chamber.

[0203] Capillary 22 is fluidly connected to secondary metering structure19. Capillary 22 is from about 0.02 mm to about 2 mm deep and has across-sectional diameter of from about 0.02 mm to about 2 mm and isconnected to secondary metering structure 19 at a position between themetering section and the overflow section. Capillary 22 is furtherfluidly connected with total hemoglobin read chamber 23. Totalhemoglobin read chamber 23 is from about 0.02 mm to about 3 cm deep, ispositioned from about 1.2 cm to about 14 cm from the center of rotation,and has a volumetric capacity of from about 5 μL to about 100 μL. Totalhemoglobin read chamber 23 is positioned radially more distal from thecenter of rotation than secondary metering structure 19, and comprises aread window translucent to light having a wavelength of from about 400nm to about 950 nm. In addition, there is no capillary or sacrificialvalving controlling fluid flow in capillary 23.

[0204] The platform also comprises control sample read cuvettes 13 and14, advantageously positioned in proximity to total hemoglobin readchamber 23. Control sample read cuvettes 13 and 14 are each from about0.02 mm to about 3 cm deep, positioned from about 1.2 cm to about 14 cmfrom the center of rotation, and have a volumetric capacity of fromabout 5 μL to about 100 μL. Control sample read cuvettes 13 and 14comprise a read window translucent to light having a wavelength of fromabout 400 nm to about 950 nm. Control sample read cuvettes 13 and 14 arenot fluidly connected to any other structure on the platform.

[0205] Air displacement channels 33 and capillary junction(s) 34, thatpermit venting of air displaced by fluid movement on the platform, arefluidly connected to the components of the platform to permit unimpededfluid flow.

[0206] As illustrated in FIGS. 12A through 12Q, in the use of thisplatform a volume of blood from about 15 μL to about 150 μL is appliedto entry port 1. Blood enters lysis subvolume 2 and lateral passageway 3under the influence of gravity and capillary forces in the absence ofrotation of the platform, as shown in FIG. 12A. Upon rotation of theplatform at a first rotational speed f₁ of from about 50 rpm to about1000 rpm, blood completely fills lysis subvolume 2 and also flowsthrough passageway 3 and into blood glucose metering chamber 4 andoverflow chamber 5, shown in FIG. 12B. Blood is retained in bloodglucose chamber 4 either due to capillary pressure or by a sacrificialvalve 6, most preferably a wax valve. Similarly, blood is retained inblood lysis subvolume 2 by a valve, most preferably a sacrificial valve7. Excess blood flows at rotational speed f₁ through overflow channel 8and into overflow chamber 9, shown in FIGS. 12C and 12D. Typical valuesfor the first rotational speed are an acceleration of about 20 to about60 rpm/sec to a final radial velocity of about 600 rpm.

[0207] After blood is metered and excess blood delivered to overflowchamber 9, the rotational speed of the disc is reduced to a rotationalspeed f_(1a) of from about 0 rpm to about 500 rpm, typically to about 60rpm, to perform a blanking measurement on sample collection cuvettearray 12, total hemoglobin read chamber 23 and blood glucose assaychamber 11. Measurements of blanking cuvettes 13 and 14 are alsoadvantageously performed.

[0208] The disc is then accelerated to a second rotational speed f₂ ofabout 200 rpm to about 2000 rpm, greater than f₁, and typically in therange of from about 800 rpm to about 1000 rpm. At this speed, capillaryvalve 6 is overcome or sacrificial valve 6 is released, and from about 1μL to about 50 μL of blood from blood glucose metering chamber 4 flowsthrough capillary 15 and into blood glucose assay chamber 11, shown inFIG. 12E. Upon entering assay chamber 11, blood fluid components areforced into absorbent matrix 10 through depression 11A. The blood fluidis incubated in matrix 10 for a time sufficient for the reagents 10A toproduce a colored product 10B in an amount proportional to the amount ofglucose in the blood fluid sample. The disc is slowed, typically to arotational speed f₄ of from about 0 rpm to about 500 rpm, typicallyabout 100 rpm, for glucose data acquisition using reflectancespectrometry; data acquisition as the disc is spinning down also enablesto instrument to set t=0 for the assay, based on a decrease inreflectance when the matrix 10 is wet by the blood fluid components andhence the matrix's scattering decreases. Development of colored product10B is shown in FIG. 12G.

[0209] The disc is then accelerated to rotational speed f₃ of about 500rpm to about 3000 rpm, typically about 1000 rpm, with release ofsacrificial valve 7A and fluid flow of from about 1 μL to about 50 μL,typically about 5 μL of blood from metered subvolume 2 through capillary7 and into blood lysis chamber 16 containing from about 25 μL to about90 μL, typically about 45 μL of blood lysis buffer. This is shown inFIG. 12H. The mixture of blood and blood lysis buffer in blood lysischamber 16 is mixed by agitation, wherein the platform is acceleratedrepeatedly from about +2000 rpm/sec to −2000 rpm/sec (wherein

+

and

−

indicate rotation in different directions), typically from about 250-500rpm/sec, over a time period of about 30 seconds to about 5 min,typically 1-2 min, as shown in FIGS. 12I and 12J.

[0210] The disc then is accelerated to a rotational speed f₅ from about200 rpm to about 2000 rpm and typically about 750 rpm, and sacrificialvalve 18 is released. Lysed blood from blood lysis chamber 16 flowsthrough capillary 17 and into secondary metering structure 19, as shownin FIG. 12K. The lysed blood solution sequentially fills throwawaysection 20, which is used as a trap for cell debris, metering section 21and excess lysed blood then fills overflow section 24. Filling ofmetering chamber 21 is immediately followed by fluid flow throughcapillary 22 and filling of total hemoglobin read chamber 23. The discis spun at rotational speed f₅ for a time sufficient to substantiallycompletely drain blood lysis chamber 16. The configuration of the bloodfluids on the disc after this spin is shown in FIG. 12L.

[0211] The disc is then accelerated to a rotational speed f_(?) fromabout 200 rpm to about 3000 rpm and typically about 750 rpm, andsacrificial valve 26 is released. A metered volume of about 1 μL toabout 50 μL, typically about 6 μL of lysed blood from metering section21 flows through capillary 27 and into boronate affinity matrix chamber28 (shown in FIG. 12M). The lysed blood solution is allowed to incubatein the chamber for a time from about 30 seconds to about 5 min,typically about 1 min, sufficient for glycated hemoglobin to bind to thematrix. This aspect of the disc is illustrated in FIG. 12N.

[0212] The disc is then accelerated to a rotational speed f_(?) fromabout 500 rpm to about 3000 rpm and typically about 1000 rpm, andsacrificial valve 32 is released. A volume of about 250 μL to about 350μL, typically about 290 μL of column wash buffer as described aboveflows from wash buffer reservoir 30 though capillary 31 and intoboronate affinity matrix chamber 28 (shown in FIG. 12O). The wash bufferdisplaces the non-glycated hemoglobin and other components of the lysedblood fluid from the affinity column matrix and into sample collectioncuvette array 12. FIGS. 12P through 12Q show sequential filling of theindividual cuvettes in sample collection cuvette array 12. The rotationspeed of the disc is reduced, to from about 0 rpm to about 500 rpm andtypically to about 60 rpm for sample collection cuvette array 12 andtotal hemoglobin read chamber 23 to be interrogatedspectrophotometrically. The glycated fraction of the blood sample isdetermine algorithmically by subtracting the non-glycated hemoglobinfraction in sample collection cuvette array 12 from the total hemoglobindetected in total hemoglobin read chamber 23.

2. Resistive Heater and Temperature Sensing Components

[0213] Temperature control elements are provided to control thetemperature of the platform. The invention provides heating elements,specifically resistive heating elements, and elements for detectingtemperature at specific positions on the platform. Heating devices arepreferably arrayed to control the temperature of the platform over aparticular and defined area, and are provided having a steep temperaturegradient with distance on the platform from the heater.

[0214] Certain resistors, including commercially-available resistiveinks (available from Dupont) exhibit a positive temperature coefficient(PTC), i.e., an increase in resistance with increasing temperature.Applying a fixed voltage across a PTC resistor screen-printed on aplastic substrate results in rapid heating, followed by self-regulationat an elevated temperature defined by the circuit design heat sink andambient temperature. In such screen-printed resistors, connection to apower source is made by first printing parallel silver conductorsfollowed by printing the PTC ink between the conductors.

[0215] A resistive heating element comprises a conductive ink connectedwith electrical contacts for activation of the heater, and resistiveinks applied between the conductive ink and in electrical contacttherewith, wherein application of a voltage (direct or alternatingcurrent) between the conductive inks results in current flow through theresistive inks and production of heat. There are two important types ofresistive inks used in the resistive heating elements of this inventionThe first is a standard polymer thick film ink, such as Dupont 7082 orDupont 7102 ink. These inks produce a surface temperature that is notself-limiting, and the temperature resulting from the use of these inksis dependent primarily on the magnitude of the applied voltage. Incontrast, the positive temperature coefficient (PTC) inks show increaseresistivity with increasing voltage, so that surface temperature isself-limiting because the amount of heat-producing current goes down asthe applied voltage goes up. PTC inks are characterized as having aparticular temperature where this self-limiting property is firstexhibited; at voltages that produce temperatures less than the criticaltemperature, the amount of heat is dependent on the magnitude of theapplied voltage.

[0216] Resistive inks useful according to the invention include Dupont7082, 7102, 7271, 7278 and 7285, and other equivalent commerciallyavailable polymer thick film ink and PTC inks.

[0217] Conductive inks useful according to the invention include Dupont5028, 5025, Acheson 423SS, 426SS and SS24890, and other equivalentcommercially available conductive inks.

[0218] Additional components of the dielectric layer that serves toinsulate the electrical circuit. Dielectric layers advantageouslycomprise dielectric inks such as Dupont 5018A. Insulation can also beachieved using pressure sensitive transfer adhesive such as 7952MP (3MCo.), or a pressure sensitive transfer adhesive deposited onto apolyester carrier layer such as 7953MP (3M Co.) or thermoplastic bondingfilms such as 3M 406, 560 or 615.

[0219] Resistive heaters of the invention are advantageously used toincubate fluids at a stable temperature and for melting sacrificialvalves as described below, and also for thermal cyclic.

[0220] Resistive and conductive inks are preferably screen-printed usingmethods and techniques well known in the art. See Gilleo, 1995, PolymerThick Film (Van Nostrand Reinhold). Inks are typically screen printed toa thickness of about 10 microns; however, repetitive screen printing ofresistive inks can be used to deposit thicker layers having reducedresistances. Both conductive and resistive inks are heat cured,typically at between 110° C. and 120° C. for about 10 minutes. Theoutline of this printing process is shown in FIG. 30. Importantly, eachof the layers must be correctly registered with one another forresistive heating to be provided. Heaters can be screen printed to anyrequired size; a minimum area for a screen-printed heater has beendetermined to be about 0.25 mm² (0.5 mm×0.5 mm).

[0221] The ability to tailor the resistance (and hence the temperatureprofile) of the resistive heaters using choice of ink formulation andreprinting of heater circuits provides control of the final electricaland thermal properties of the resistive heating elements of theinvention. The resistance can also be controlled through connection ofseries and parallel configurations of resistive elements.

3. Sacrificial Valves

[0222] The ability to specifically generate heat at a particularlocation on a Microsystems platform of the invention also enables theuse of sacrificial valves that can be released or dissolved using heat.For the purposes of this invention, the term

sacrificial valve

is intended to encompass materials comprising waxes, plastics, and othermaterial that can form a solid or semi-solid fluid-tight obstruction ina microchannel, capillary, chamber, reservoir or other microfluidicscomponent of the platforms of the invention, and that can be melted ordeformed to remove the obstruction with the application of heat.Sacrificial valves are preferably made of a fungible material that canbe removed from the fluid flow path. In preferred embodiments, saidsacrificial valves are wax valves and are removed from the fluid flowpath by heating, using any of a variety of heating means includinginfrared illumination and most preferably by activation of resistiveheating elements on or embedded in the platform surface as describedherein. For the purposes of this invention, the term

wax

is intended to encompass any solid, semi-solid or viscous liquidhydrocarbon, or a plastic. Examples include mondisperse hydrocarbonssuch as eicosane, tetracosane and octasone, and polydispersehydrocarbons such as paraffin. In the use of wax sacrificial valves,application of a temperature higher than the melting temperature of thewax melts the valve and removes the occlusion from the microchannel,capillary or other fluidic component of the microsystems platforms ofthe invention. Particularly when the sacrificial valve is melted on arotating Microsystems platform of the invention, the melted wax to flowthrough the microchannel, capillary or other fluidic component of theMicrosystems platforms of the invention and away from the original siteof the valve.

[0223] One drawback, however, is the possibility that the wax willrecrystallize as it flows away from the original valve site, andconcomitantly, away from the localized heat source. Recrystallizationresults in re-occlusion of the microchannel, capillary or other fluidiccomponent of the Microsystems platforms of the invention, potentiallyand most likely at a site other than the site of a localized heatsource, and therefore likely to foul fluid movement on the disc. Onesolution for this problem is the inclusion in the sacrificial wax valvesof the invention of a wax recrystallization chamber positioned

downstream

from the position of the wax valve. Preferably, the waxrecrystallization chamber is fluidly connected with the microchannel,capillary or other fluidic component of the Microsystems platforms ofthe invention that was occluded by the wax sacrificial valve. Typically,the wax recrystallization chamber is a widening of the microchannel,capillary or other fluidic component of the Microsystems platforms ofthe invention so that recrystallized wax can harden on the walls of themicrochannel, capillary or other fluidic component of the Microsystemsplatforms of the invention with enough distance between said walls thatthe recrystallized wax does not re-occlude the microchannel, capillaryor other fluidic component of the Microsystems platforms of theinvention. Preferably, the heating element, most preferably theresistive heating element of the invention, extends past the site of thewax valve and overlaps at least a portion of the wax recrystallizationchamber, thereby retarding the propensity of the wax valve torecrystallize.

[0224] It is also recognized that this propensity of wax valves torecrystallize can be exploited to create a wax valve at a particularlocation in a microchannel, capillary or other fluidic component of themicrosystems platforms of the invention. In this embodiment, aparticular location can be kept below a threshold temperature by failingto apply heat at that location, and a wax valve material can bemobilized from a storage area on a platform by heating and them allowedto flow under centripetal acceleration to a particularly

cold

site where a wax valve is desired. An advantage of wax valves in thisregard is that the proper positioning an activation of resistive heaterelements enables flexibility in choosing when and whether a particularmicrochannel, capillary or other fluidic component of the Microsystemsplatforms of the invention is to be occluded by a wax sacrificial valve.

[0225] In particularly preferred embodiments, the sacrificial valves ofthe invention comprise a cross-linked polymer that displays thermalrecover, most preferably a cross-linked, prestressed, semicrystallinepolymer; an example of a commercially available embodiment of such apolymer is heat recoverable tubing (#FP301H, 3M Co., Minneapolis,Minn.). Using these materials, at a temperature less than the

melting

temperature (T_(m)), the polymer occludes a microchannel, capillary orother fluidic component of the Microsystems platforms of the invention.At a temperature greater than T_(m),, however, the polymer reverts toits pre-stressed dimensions by shrinking. Such shrinking is accompaniedby release of the occlusion from the microchannel, capillary or otherfluidic component of the microsystems platforms of the invention. Suchembodiments are particular preferred because the polymer remains in situand does not recrystallize or otherwise re-occlude the microchannel,capillary or other fluidic component of the microsystems platforms ofthe invention. Also, such embodiments do not require the more extensivemanipulation in preparing the platforms of the invention that wax valvesrequire.

[0226] In another embodiment, the sacrificial valves of the inventioncomprise a thin polymeric layer or barrier dividing twoliquid-containing microchannel, capillary or other fluidic component ofthe Microsystems platforms of the invention, that can burst whensufficient temperature and/or pressure is applied.

[0227] Another embodiment of the sacrificial valves of the invention areprovided wherein a screen-printed resistive heater element is itself avalve. In this embodiment, the resistive heater element isscreen-printed on a substrate such as polyester that divides twoliquid-containing microchannel, capillary or other fluidic component ofthe Microsystems platforms of the invention. In these embodiments,localized application of heat using a resistive heating element is usedto melt the substrate dividing the liquid-containing microchannel,capillary or other fluidic component of the microsystems platforms ofthe invention. Preferably, in this embodiment the two liquid-containingmicrochannel, capillary or other fluidic component of the microsystemsplatforms of the invention are positioned in adjacent layers through thevertical thickness of the platform.

[0228] As described above, the screen-printed resistive heater elementsof this invention provide localized application of heat to amicrosystems platform. The degree of localization achieved using theseresistive heating elements is sufficient to provide for the placement oftwo adjacent sacrificial valves separated by a distance of 0.15 cm.

4. Detectors and Sensors

[0229] Detection systems for use on the microsystem platforms of theinvention include spectroscopic, electrochemical, physical, lightscattering, radioactive, and mass spectroscopic detectors. Spectroscopicmethods using these detectors encompass electronic spectroscopy(ultraviolet and visible light absorbance, fluorescence, luminescence,and refractive index), vibrational spectroscopy (IR and Raman), andx-ray spectroscopies (x-ray fluorescence and conventional x-ray analysisusing micromachined field emitters, such as those developed by the NASAJet Propulsion Lab, Pasadena, Calif.).

[0230] General classes of detection and representative examples of eachfor use with the microsystem platforms of the invention are describedbelow. In addition, the detection implementation systems utilizing thedetectors of the invention can be external to the platform, adjacent toit or integral to the disk platform.

Spectroscopic Methods 1. Fluorescence

[0231] Fluorescence detector systems developed for macroscopic uses areknown in the prior art and are adapted for use with the microsystemplatforms of this invention. For example, an excitation source such as alaser is focused on an optically-transparent section of the disk. Lightfrom any analytically-useful portion of the electromagnetic spectrum canbe coupled with a disk material that is specifically transparent tolight of a particular wavelength, permitting spectral properties of thelight to be determined by the product or reagent occupying the reservoirinterrogated by illumination with light. Alternatively, the selection oflight at a particular wavelength can be paired with a material havinggeometries and refractive index properties resulting in total internalreflection of the illuminating light. This enables either detection ofmaterial on the surface of the disk through evanescent lightpropagation, or multiple reflections through the sample itself, whichincreases the path length considerably.

[0232] Alternative configurations appropriate for evanescent wavesystems are provided as understood in the art (see Glass et al., 1987,Appl. Optics 26: 2181-2187). Fluorescence is coupled back into awaveguide on the disk, thereby increasing the efficiency of detection.In these embodiments, the optical component preceding the detector caninclude a dispersive element to permit spectral resolution. Fluorescenceexcitation can also be increased through multiple reflections fromsurfaces in the device whenever noise does not scale with path length inthe same way as with signal.

[0233] In another type of fluorescence detection configuration, light ofboth the fluorescence excitation wavelength and the emitted lightwavelength are guided through one face of the device. An angle of 90degrees is used to separate the excitation and collection opticaltrains. It is also possible to use other angles, including 0 degrees,whereby the excitation and emitted light travels colinearly. As long asthe source light can be distinguished from the fluorescence signal, anyoptical geometry can be used. Optical windows suitable for spectroscopicmeasurement and transparent to the wavelengths used are included atappropriate positions (i.e., in “read” reservoir embodiments ofdetecting chambers) on the disk. The use of this type of fluorescence inmacroscopic systems has been disclosed by Haab et al. (1995, Anal. Chem.67: 3253-3260).

2. Absorbance Detection

[0234] Absorbance measurements can be used to detect any analyte thatchanges the intensity of transmitted light by specifically absorbingenergy (direct absorbance) or by changing the absorbance of anothercomponent in the system (indirect absorbance). Optical path geometry isdesigned to ensure that the absorbance detector is focused on a lightpath receiving the maximum amount of transmitted light from theilluminated sample. Both the light source and the detector can bepositioned external to the disk, adjacent to the disk and moved insynchrony with it, or integral to the disk itself. The sample chamber onthe disk can constitute a cuvette that is illuminated and transmittedlight detected in a single pass or in multiple passes, particularly whenused with a stroboscopic light signal that illuminates the detectionchamber t a frequency equal to the frequency of rotation or multiplesthereof. Alternatively, the sample chamber can be a planar waveguide,wherein the analyte interacts on the face of the waveguide and lightabsorbance is the result of attenuated total internal reflection (i.e.,the analyte reduces the intensity source light if the analyte issequestered at the surface of the sample chamber, using, for example,specific binding to a compound embedded or attached to the chambersurface; see Dessy, 1989, Anal. Chem. 61: 2191).

[0235] Indirect absorbance can be used with the same optical design. Forindirect absorbance measurements, the analyte does not absorb the sourcelight; instead, a drop in absorbance of a secondary material is measuredas the analyte displaces it in the sample chamber. Increasedtransmittance therefore corresponds to analyte concentration.

3. Light Scattering

[0236] Turbidity can also be measured on the disk. Optics are configuredas with absorbance measurements. In this analysis, the intensity of thetransmitted light is related to the concentration of the light-scatteredparticles in a sample. An example of an application of this type ofdetection method is a particle agglutination assay. Larger particlessediment in a rotating disk more rapidly than smaller particles, and theturbidity of a solution in the sample chamber before and after spinningthe disk can be related to the size of the particles in the chamber. Ifsmall particles are induced to aggregate only in the presence of ananalyte, then turbidity measurements can be used to specifically detectthe presence of an analyte in the sample chamber. For example, smallparticles can be coated with an antibody to an analyte, resulting inaggregation of the particles in the presence of the analyte as antibodyfrom more than one particle bind to the analyte. When the disk is spunafter this interaction occurs, sample chambers containing analyte willbe less turbid that sample chambers not containing analyte. This systemcan be calibrated with standard amounts of analyte to provide a gauge ofanalyte concentration related to the turbidity of the sample under a setof standardized conditions.

[0237] Other types of light scattering detection methods are providedfor use with the Microsystems platforms and devices of the invention.Monochromatic light from a light source, advantageously a laser lightsource, is directed across the cross-sectional area of a flow channel onthe disk. Light scattered by particles in a sample, such as cells, iscollected at several angles over the illuminated portion of the channel(see Rosenzweig et al., 1994, Anal. Chem. 66: 1771-1776). Data reductionis optimally programmed directly into the device based on standards suchas appropriately-sized beads to relate the signal into interpretableresults. Using a calibrated set of such beads, fine discriminationbetween particles of different sizes can be obtained. Anotherapplication for this system is flow cytometry, cell counting, cellsorting and cellular biological analysis and testing, includingchemotherapeutic sensitivity and toxicology.

Analytic Methods

[0238] It will be understood that the interpretation of the opticaldetection data from performance of analytical assays as provided by theinvention may require transformation of “raw” data into informationuseful for the operator. For example, “reflectance” is determined as thedifference between the “signal” and the “dark signal” (i.e., the signaldetected in the absence of transmitted light), normalized by thedifference between the “reference” and the “dark reference,” whereineach of the reference values is determined for a “blank” cuvette notcontaining a sample. Similarly, the detection methods used by theinvention also include methods for eliminating artifactual signalproduced by interfering absorbing or light-scattering components, suchas scratches or particulate matter. Advantageously, normalized readingsare collected at a wavelength greater than 650 nm, more preferably660±10 nm as a reference. This reading is then subtracted from thesample reading to as a correction thereof.

[0239] Absorbance from certain components of biological fluid sample,such as hemoglobin in blood, are advantageously removed by using a lightsource with a wavelength at the absorbance peak of the interferingmaterial; for hemoglobin, this is about 425 nm. In preferredembodiments, light of this wavelength is provided by a bluelight-emitting diode (LED) having a wavelength of about 430 nm. Thelight emission profile of the LED was found to overlap sufficiently withthe hemoglobin absorbance profile to effectively quench the hemoglobinabsorbance signal in the glucose assays of the invention. The glucosesignal was then further treated analytically by subtracting a factortimes the (blue-red) signal from the (orange-red) signal, wherein the(orange) signal was the wavelength specific for the glucose assayproduct. The factor used is dependent on the optics, the reading chamberstructure, spectral properties of the light sources and filters, and thespectral absorbance characteristics of hemoglobin and the coloredproduct of the glucose oxidase reaction. The factor can be determinedempirically using solutions of known glucose and hemoglobinconcentrations, both singly and in combination.

[0240] It will be understood in the art that similar combinations oflight sources and interrogated wavelengths can be advantageously used toreduce or eliminate the contribution of other interfering species inoptical detection methods used according to the invention.

5. Chemistries

[0241] As described above with regard to the microfluidic components ofthe Microsystems platforms of the invention, the present inventionprovides platforms for performing chemical, biochemical, enzymatic,immunological and other assays on fluid samples, most preferably whereinthe fluid sample is a biological fluid sample.

[0242] Two exemplary types of assay formats are explicitly set forthherein; one of ordinary skill will recognize that the disclosure isgenerally applicable to a variety of assay systems as set forth, forexample, in CLINICAL GUIDE TO LABORATORY TESTS, Tietz, ed., W. B.Sanders Co: Philadelphia, 1995. A representative and non-limiting sampleof assays advantageously performed using the microsystems platforms ofthe invention are set forth in Table I below. TABLE I Assay for:Analyte/Detection Type Components on solid phase: Acid Phosphataseenzyme/colorimetric Alpha-naphthol phosphate, Fast Red TR dye¹ Alanineenzyme/colorimetric Alanine, alpha-ketoglutarate, 2,4- Aminotransferasedinitrophenylhadrazine dye Albumin protein/colorimetric Bromcresol greendye Alkaline Phosphatase enzyme/colorimetric p-nitrophenyl phosphateAmylase enzyme/colorimetric4,6-ethylidine(G7)-p-nitrophenyl(G1)-alpha,D- maltoheptaside,alpha-glucosidase Apolipoprotein A-1 lipoprotein/immunoturbidimetricAnti-ApoA1 antibody, polyethylene glycol² Direct Bilirubin organiccompound/colorimetric Diazotized sulfanilic acid Total Bilirubin organiccompound/colorimetric Caffeine, benzoate, acetate, diazotized sulfanilicacid Calcium mineral/colorimetric Cresolphthalein complexone Cholesterollipid/colorimetric Cholesterol esterase, cholesterol oxidase, 4-aminoantipyrine, p-hydroxybenzene sulfonate Fructosamine glycated serumNitroblue tetrazolium protein/colorimetric Gamma-glutamylenzyme/colorimetric L-gamma-glutamyl-3-carboxy-4-nitroanilide,transferase glycylglycine Iron mineral/colorimetric Ferrozine, reducingagent Microprotein (urine protein/colorimetric Pyrogallol red-molybdatecomplex or CSF) Urea organic compound/colorimetric Diacetylmonoxime,heat Specific Protein immunologically reactive Antibody reactive withunique species, etc, site; species, subspecies orproteins/immunoturbidimetry precipitation enhancers (e.g., anti-alpha-1-variant antitrypsin IgG, polyethylene glycol)³ Antibodies againstimmunoreaction to bacteria or Immunoreactive component from infectiveinfectious agents virus infection/heterogeneous agent, enzyme linked toantibody against the enzyme immunoassay primary immunoglobulin speciesof reaction to the agent, enzyme substrate linked to color generation(e.g., Epstein-Barr early antigen, anti-human IgG conjugated tohorseradish peroxidase, 3-3′-5-5′-tetramethylbenzidine) Drugsimmunologically reactive Competition between drug and drug-enzymetherapeutic drugs or drugs of reagent for anti-drug antibody bindingsites abuse/homogeneous enzyme where antibody binding inhibits enzymeimmunoassay activity, enzyme substrate linked to color generation (e.g.,phenobarbital-glucose-6- phosphatase, anti-phenobarbital IgG, NAD)⁴

[0243] The requirements for performing such assays on the solid phase(such as the matrices disclosed herein) include: the ability to link, byone or a series of chemical reaction steps, the presence of an analyteof interest in a fluid sample, most preferably a biological fluidsample, to the quantitative generation of a product detectable byoptical methods as disclosed herein; stabilization of the necessaryreagents (chemicals or biochemicals) onto the solid phase so that thereagents retain their potencies or activities.

[0244] A second general scheme for performing assays on the Microsystemsplatforms of the invention involve miniaturized versions of affinitychromatography column separations, wherein the analyte specificallybinds to a material in a chamber or on a surface, most preferably aderivatized surface, of the platform, or is bound to a material such asa bead, chromatography resin, or membrane on the surface of theplatform, so that the remainder of the fluid sample can be washed fromthe affinity matrix and the analyte separated thereby. In certainpreferred embodiments, the analyte is detected indirectly, wherein thebiological fluid sample is interrogated after passage of the sample overthe chromatograohy matrix. Such detection methods can be subtractive,wherein the interrogated optical property of the biological fluid sampleafter passage over the chromatography matrix is compared with the sameproperty of a portion of the sample that has not been passed over thematrix; or directly, wherein the analyte is dissociated from thechromatography matrix (either non-specifically, using for example a saltor dielectric gradient, or specifically, using a binding competitor thatdisplaces the analyte from the chromatography matrix).

[0245] Examples of analytes advantageously separated from biologicalfluid samples and the column affinity material(s) used therefore are setforth in Table II. TABLE II Column Affinity Material Bound SpeciesDiatomateous earth DNA Protein A-agarose IgG Heparin-agarose coagulationproteins, Protein C, growth factors, lipoproteins, steroid receptorsBlue agarose albumin, coagulation factors, interferon, enzymes requiringcofactors with adenyl group Streptavidin-agarose biotinylated moleculesCon A-agarose glycoproteins, polysaccharides with terminal mannose orglucose Lentil lectin-agarose glycoproteins, polysaccharides withbranched mannose with fucose linked α(1,6) to N-acetyl-glucosamine Wheatgern lectin-agarose glycoproteins, polysaccharides with chitobiose coreof N-linked oligosaccharides Peanut lectin-agarose glycoproteins,polysaccharides with terminal β-galactose Arginine-agarose serineproteases Calmodulin-agarose ATPases, phosphodiesterases,neurotransmitters, protein kinases Gelatin-agarose FibronectinGlutathione-agarose S-transferases, glutathione-dependent proteinsLysine-agarose plasminogen, plasminogen activator, ribosomal RNA DNA(denatured)-agarose DNA polymerase, RNA polymerase, T4 polynucleotidekinase, exonuclease, deoxyribonucleases DNA (native)-celluloseGlucocorticoid receptor, DNA polymerase, DNA binding proteins 2′5′ADP-agarose NADP-dependent dehydrogenases 5′ AMP-agarose NAD-dependentdehydrogenases, ATP-dependent kinases 7-Methyl-GTP-agarose eukaryoticmRNA, cap-binding protein Poly(U)-agarose mRNA, reverse transcriptase,interferon, plant nucleic acids C8-silica Proteins

[0246] It will be appreciated that preparative embodiments of theaffinity chromatographic column separations are within the scope of theinvention.

[0247] The following Examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.

EXAMPLE 1 Blood Glucose Assay Fluidics Structure

[0248] A microsystems platform provided by the invention andspecifically designed for performing blood glucose assay is illustratedin FIG. 1. Disk embodiments of the platforms of the invention werefashioned from machined acrylic. The overall disc dimensions include anouter radius of about 6 cm and an inner radius of about 0.75 cm, whereinthe disk was mounted on the spindle of a rotary device. The thickness ofthe fluidics disc was 3.2 mm, which was mounted on a conventionalcompact disc (CD) having a thickness of 1.2 mm. All surfaces coming intocontact with blood on the platform are advantageously treated withheparin, EDTA or other anticoagulants to facilitate fluid flowthereupon.

[0249] The components of the blood glucose assay were prepared asfollows. Blood sample entry port chamber 101 having a depth in theplatform surface of about 0.32 cm and lateral dimensions of about 1 cmwas constructed on the platform, and designed to accommodate a volume of100 μL. This entry port was fluidly connected with a metering capillary102 having a square cross-sectional diameter of about 0.1 cm deep×0.5 cmwideand proximal ends rounded with respect to entry port 101; the lengthof this metering capillary array was sufficient to contain a totalvolume of about 16 μL. The entry port was also fluidly connected with anoverflow capillary 103 having a cross-sectional diameter of about 0.05cm×0.075 cm and proximal ends rounded with respect to entry port 101.The overflow capillary was fluidly connected with a two-layered overflowchamber 105 having a first depth in the platform of about 0.025 cm and asecond depth in the platform of about 0.25 cm, greater than the depth ofthe overflow capillary 103. Metering capillary 102 was fluidly connectedto metered blood fluid chamber 104 having a depth in the platformsurface of 1 mm and greater than the depth of the metering capillary102. Each of the overflow and fluid chambers was also connected with airports or air channels, such as 114, that have dimensions of 0.025 cmdeep and permitted venting of air displaced by fluid movement on theplatform. A capillary junction 115 that was 0.051 cm deep was present inthe air channel to prevent fluid flow into the air channel.

[0250] Entry port 101 was positioned on the platform about 2 cm from thecenter of rotation. Metering chamber 102 extended about 1 cm from entryport 101. The extent of the length of overflow capillary 103 was 300%greater than the extent of the length of metering capillary 102. Theposition of blood fluid chamber 104 was about 4.6 cm from the center ofrotation, and the position of overflow chamber 105 was about 4 cm fromthe axis of rotation.

[0251] Blood fluid chamber 104 acted as a capillary barrier thatprevented fluid flow from metering chamber 102 at a first, non-zerorotational speed f₁ of about 3000 rpm that was sufficient to permitfluid flow comprising overflow from the entry port 101 through overflowcapillary 103 and into overflow chamber 105. This capillary boundary wasconstructed to be overcome at a second rotational speed f₂ of about 600rpm (so that f₂>f₁). Blood fluid chamber 104 was fluidly connected tocapillary 110 that was about 0.025 cm deep and had a cross-sectionaldiameter of about 0.025 cm and was connected to capillary or sacrificialvalve 111. Sacrificial valve 111 was further fluidly connected withcapillary 112 that was about 0.25 mm deep and had a cross-sectionaldiameter of about 0.05 cm, and capillary 112 was fluidly connected toassay chamber 107. Sacrificial valve chamber 111 was positioned tosequester melted wax produced by release of the sacrificial valve.

[0252] Assay chamber 107 comprised a depression in the surface of theplatform having a depth of about 0.1 cm, and further comprised acircular or rectangular concave depression 113 connected to capillary112. Assay chamber 107 also comprised a pad or matrix 106 comprising apositively-charged nylon matrix having a pore size of about 0.8 μm. Thepore size of matrix 106 was chosen to inhibit or prevent blood cellentry into the matrix. The matrix was positioned in assay chamber 107 tobe in fluidic contact with depression 113, covering depression 113 andhaving a surface area greater than the surface area of depression 113.The matrix was impregnated with immobilized reagents 108 which produce adetectable product proportional to the concentration of glucose in ablood sample. The detectable product was a colored product 109, i.e., aproduct absorbing light at a detectable, visible wavelength.

[0253] As illustrated in FIGS. 2A through 2E, in the use of thisplatform an imprecise volume (about 30 μL of fluid) of blood was appliedto the entry port 101. The fluid wicked into air channel 114 and wasstopped by capillary junction 115. Fluid also wicked into meteringcapillary 102 and overflow capillary 103. Fluid flowed through themetering capillary 102 and overflow capillary 103 at no rotational speeduntil the fluid reached capillary junctions at the junction betweenmetering chamber 102 and blood fluid chamber 104 and overflow capillary103 and overflow chamber 105. Metering capillary 102 was constructed todefine a precise volume of about 15 μL of blood between entry port 101and the capillary junction at fluid chamber 104, which was designed tobe at least the amount of the fluid placed by the user in entry port101.

[0254] After sample loading by a user and filling of metering chamber102 and overflow capillary 103 at zero rotational speed, the platformwas spun at a first rotational speed f₁ of about 300 rpm, which wassufficient to motivate fluid flow through the overflow capillary 103 inthis microfluidics array, wherein entry port 101 had a depth of about0.3 cm, metering chamber 102 had dimensions of about 0.1 cm deep×0.4 cmwide in cross-section and about 0.5 cm in length from the center ofrotation, and overflow capillary 103 had dimensions of about 0.05cm×0.075 cm in cross-section and about 2.7 cm in length from the centerof rotation.

[0255] Due to the greater distance from the center of rotation of theend of overflow capillary 103 than the end of metering chamber 102, atrotational speed f₁ fluid flowed through overflow capillary 103 intooverflow chamber 105. The platform was spun until all excess fluid wasevacuated from entry port 101 and into overflow chamber 105, except thefluid contained in metering capillary 102.

[0256] At a second rotational speed f₂ of about 1000 rpm, the preciseamount of fluid (16 μL) contained in metering capillary 102 wasdelivered into fluid chamber 104. Fluid movement into fluid chamber 104was accompanied by filling of capillary 110.

[0257] In embodiments comprising a sacrificial valve 111 in-line withcapillary 110 at a position between capillary 110 and 112 shown in FIG.2A, release of the sacrificial valve resulted in fluid flow throughcapillary 112 and into assay chamber 107. In said embodiments, fluidflow was achieved at rotational speed f₂ with removal of the sacrificialvalve. In embodiments of the platforms of the invention comprisingcapillary valve 111 at a position between capillary 110 and 112 shown inFIG. 2B, capillary 110 filled along with filling of blood fluid chamber104 until blood reached capillary junction 111 at the junction betweencapillary 110 and capillary 112; in such embodiments, the capillaryjunction had a depth of about 0.05 cm. At a third rotational speed f₃ ofabout 500 rpm, the fluid contained in blood fluid chamber 104 wasdelivered into assay chamber 107 (FIG. 2B). Blood flowing into assaychamber 107 was preferentially directed to depression 113 in the assaychamber; the dimensions of depression 113 are conveniently chosen to beable to contain substantially all of the blood fluid of the samplemetered through metering chamber 102 into assay chamber 107 (FIG. 2C).Displaced air flows through air channel 114, and was vented to thesurface of the disc.

[0258] As blood flowed into depression 113, the fluid component of theblood is driven by pressure and hydrophilic forces into matrix 106,comprising a positively-charged nylon matrix having a pore size of about0.8 μm; this pore size was chosen to prevent the cellular components ofthe blood from entering the matrix (FIG. 2D). The cellular bloodcomponents were retained in depression 113 and the fluid component wasefficiently distributed into matrix 106. As the fluid component of theblood entered matrix 106, dried reagents 108 were solubilized and thereaction of the blood component catalyzed by said reagents proceeded.This reaction(s) went to completion within about 1 min. Reaction of theblood component(s) with reagents 108 produce colored product 109 whichwas then detected (FIG. 2E), as described below in Example 4.

EXAMPLE 2 Glycated Hemoglobin Assay—Fluidics Structure

[0259] A Microsystems platform provided by the invention andspecifically designed for performing a glycated hemoglobin assay isillustrated in FIG. 11.

[0260] Construction of the disk embodiments of the platforms of theinvention are as described in Example 1. The blood application andmetering components and their dimensions and relationships to oneanother are identical to those described above, comprising sample entryport chamber 801, metering capillary 802, overflow capillary 803,metered blood fluid chamber 804 and overflow chamber 805. As in Example1, each of the overflow and fluid chambers is also connected with airports or air channels, such as 814, and capillary junction(s) 815, thatpermit venting of air displaced by fluid movement on the platform.

[0261] Blood fluid chamber 804 was fluidly connected to capillary 810that was about 0.25 mm deep and had a cross-sectional diameter of about0.25 mm and was connected to sacrificial valve 811. Sacrificial valve811 was further fluidly connected with capillary 812 that was about 0.25mm deep and had a cross-sectional diameter of about 0.25 mm. Capillary812 was further fluidly connected to mixing chamber 815 that was about0.25 cmcm deep, had a cross-sectional diameter of about 1 cm, and waspositioned about 2.5 cm from the center of rotation. Lysis buffer wasloaded directly onto mixing chamber 815 in this embodiment and did notuse capillary 818 or lysis buffer chamber 816 as shown in the Figure. 45μL of lysis buffer was applied to the mixing chamber as a solution of0.1% Triton X100 in 50 mM Tris pH 9.5.

[0262] Mixing chamber 815 was fluidly connected to capillary 817 thatwas about 0.25 mm deep and had a cross-sectional diameter of about 0.25mm, and was connected to secondary metering structure 819. Secondarymetering structure 819 was about 0.1 cm deep and was positioned about3.5 cm from the center of rotation. Secondary metering structure 819 wasconstructed to comprise two sections. A metering section was arrangedproximal to the entry position of capillary 817 and was separated froman overflow section by a septum that extended from the distal wall ofthe structure to a position just short of the proximal wall of thestructure. This arrangement produced a fluid connection between thefirst metering section having a volumetric capacity of about 6.4μL andthe overflow section having an excess volumetric capacity of 90 μL. Thevolumetric capacity of the overflow section was sufficient toaccommodate the largest blood fluid volume applied to the disk.

[0263] Capillary 821 was in fluid connection with secondary meteringstructure 819 at the distal wall of the metering section. Capillary 821was about 0.15 cm deep and had a cross-sectional diameter of about 0.05cm and was connected to boronate affinity matrix chamber 822. Boronateaffinity matrix chamber 822 was about 0.15 cm deep, had across-sectional diameter of about 0.3 cm and was positioned about 4.8 cmfrom the axis of rotation. Boronate affinity matrix chamber 822 wasfilled with boronate-functionalized agarose beads having a mean diameterof about 60 μm; the beads were maintained in the chamber 822 using aporous frit 827. Fluid flow through capillary 821 was connected tocapillary or sacrificial valve 823. Boronate affinity matrix chamber 822was further comprised of a translucent window that permitted reflectivespectrophotometry of the contents of the chamber.

[0264] Boronate affinity matrix chamber 822 was further fluidlyconnected to capillary 828. Capillary 828 was about 0.25 mm deep and hada cross-sectional diameter of about 0.25 mmand was connected to columnwash buffer reservoir 829. Column wash buffer reservoir 829 was about0.25 cm deep and had a cross-sectional diameter of about 2 cm and waspositioned about 3.6 cm from the axis of rotation, more proximal to theaxis of rotation than boronate affinity matrix chamber 822. Column washbuffer reservoir 829 comprises 290 μL of column preparation buffer thatwas a solution of magnesium chloride, taurine, D,L-methionine, sodiumhydroxide, antibiotics and stabilizers constituted according to themanufacturer's instructions (Isolab Inc. #SG-6220). Fluid flow throughcapillary 828 was connected to capillary or sacrificial valve 836.

[0265] Boronate affinity matrix chamber 822 was further fluidlyconnected to capillary 832. Capillary 832 was about 0.5 m deep and had across-sectional diameter of about 0.5 mm and was connected tonon-glycated hemoglobin read chamber 834. Chamber 834 was about 0.25 cmdeep and had a cross-sectional diameter of about 2 cm and was positionedabout 5 cm from the axis of rotation and was further comprised of atranslucent window that permitted reflective spectrophotometry of thecontents of the chamber at 430 nm.

[0266] As illustrated in FIG. 1, in the use of this platform about a 6.4μL volume of blood was applied to blood fluid chamber 804, eitherdirectly or using the metering components of the platform describedabove. Blood flowing through capillary 810 and lysis buffer contained inmixing chamber 815 were mixed in the mixing chamber. A 45 μL volume oflysis buffer was mixed with the blood sample. Fluid flow within mixingchamber 815 was turbulent, in contrast to fluid flow through capillaries810 or 818, which was primarily laminar, so that mixing occurredpredominantly in mixing chamber 815. Fluid flow proceeded throughchannel 817 and into secondary metering structure 819. The mixture oflysis buffer and blood, comprising a lysed blood sample 841, flowed at arotational speed f_(?) of about 750 rpm into secondary meteringstructure 819 with release of a sacrificial valve 853. Lysed bloodsample 841 entered and filled the metering section of secondary meteringstructure 819. Any additional lysed blood sample then emptied into theoverflow chamber of secondary metering structure 819 and filled thetotal hemoglobin read chamber. Most preferably, a sufficient volume oflysis buffer and blood sample was applied to the disc to fill at leastthe metering sections of secondary metering structure 819 and the totalhemoglobin read chamber.

[0267] After the lysed blood sample 841 was completely transferred tosecondary metering structure 819, capillary or sacrificial valve 823 wasreleased, allowing the metered lysed blood sample from the meteringsection of secondary metering structure 819 through capillary 821 andinto boronate affinity matrix 822. Capillary or sacrificial valve 836was then released, allowing about 290 μL of column wash buffer 843 toflow at rotational speed f_(?) through capillary 830 and into boronateaffinity matrix 822. Continued or discontinuous rotation motivatescolumn preparation buffer through boronate affinity matrix 822 and intonon-glycated hemoglobin read chamber 834. The control sample readcuvettes 8− and 8˜ were then then illuminated by light at a wavelengthof 430 nm and the blank reading, i.e. reflectance from cuvettescontaining only buffer, was determined. The non-glycated hemoglobin readwindow was then illuminated by light at a wavelength of 430 nm and theconcentration of non-glycated hemoglobin in the sample that has elutedfrom the column was determined by reflectance spectroscopy. The totalhemoglobin read window was also illuminated by light at a wavelength of430 nm and the concentration of total hemoglobin in the lysed sample wasdetermined by reflectance spectroscopy.

[0268] The amount of non-glycated hemoglobin in the sample is determinedby dividing the amount of hemoglobin obtained by illuminating the elutedfraction by the amount of hemoglobin obtained in the total fraction.

EXAMPLE 3 Combination Glucose Concentration—Glycated Hemoglobin AssayPlatform

[0269] A microsystems platform provided by the invention andspecifically designed for performing both a determination of bloodglucose concentration and a glycated hemoglobin assay is illustrated inFIGS. 12A through 12Q and 13A through 13E.

[0270] Construction of the disk embodiments of the platforms of theinvention were as described above. FIG. 13B shows a detailed descriptionof the microfluidics components of the platform, which are described inadditional detail below. FIG. 13C shows the geometry of a screen printedelectrical lead layer deposited on a mylar substrate. FIG. 13D shows thepositions of screen printed heaters activated by the electrical leads ofthe lead layer and screen printed on mylar. FIG. 13E shows a overlay ofthese components in the assembled disc.

[0271] Referring to the microfluidics components of the platform shownin FIG. 13B, an entry port 1 is positioned on the top surface of thedisc and is open for the user to apply an unmetered sample. Entry port 1is about 0.32 cm deep, has a cross-sectional diameter of about 1 cm, ispositioned about 2 cm from the center of rotation, and is fluidlyconnected to capillary channel 1A that is about ‘1 mm deep and has across-sectional diameter of about 1 mm. Capillary channel 1A is fluidlyconnected to metering component 2, which comprises four sections. Thefirst section is rectangularly-shaped and extends in a directionproximal to the axis of rotation away from its fluid connection withcapillary channel 1A. About half way up this rectangular section is alateral chamber 3, which empties into a blood glucose metering chamber 4and an overflow chamber 5. The first section of the metering componentis about 0.1 cm deep, has a cross-sectional diameter of about 0.4 cm, ispositioned about 2.2 cm from the center of rotation, and has avolumetric capacity of about 5 microliters for Hb assay. The lateralchamber 3 is 1 mm deep, has a cross-sectional diameter of 1 mm, and ispositioned 2.3 cm from the center of rotation. Blood glucose meteringchamber 4 is 1 mm deep, has a cross-sectional diameter of 4.5 mm, ispositioned 2.6 cm from the center of rotation and has a volumetriccapacity of 16 μL. Overflow chamber 5 is 1 mm deep, has across-sectional diameter of 2 mm, is positioned 2.6 cm from the centerof rotation and has a volumetric capacity of 7 μL.

[0272] Overflow chamber 5 is fluidly connected to overflow channel 8that is 0.75 mm deep, has a cross-sectional diameter of 0.75 mm andextends 3.8 cm from overflow chamber 5. Overflow channel 5 is fluidlyconnected to short sample detection cuvette 9 that is two depths: 0.25mm and 2.2 mm(inner) deep, has a cross-sectional diameter of 2.5 mm, ispositioned 4-5.8 cm from the center of rotation and has a volumetriccapacity of 50 μL.

[0273] Blood glucose metering chamber 4 is fluidly connected tocapillary 15 that is about 0.25 mm deep, has a cross-sectional diameterof about 0.25 mm and extends about 0.1 cm from blood glucose meteringchamber 4. Capillary 15 is connected to sacrificial wax valve 6, whichis further fluidly connected with wax recrystallization chamber 6A thatis about 0.5 mm deep and has a volumetric capacity sufficient tosequester melted wax from a released wax valve and prevent occlusion ofthe lumen of the capillary 15 controlled by the valve. Capillary 15 isfurther fluidly connected with glucose assay chamber 11 that is about0.1 cm deep, has a cross-sectional diameter of about 0.5 mm, ispositioned about 5 cm from the center of rotation and has a volumetriccapacity of about 10 μL. Glucose assay chamber 11 comprises a depression11A in the surface of the platform having a depth of about 0.1 cm, mostpreferably comprising a circular or rectangular depression connected tocapillary 15 so that blood flows into the chamber through the bottom ofdepression 11A. Depression 11A is constructed to have a volumetriccapacity of from half to twice the assay volume. Blood glucose assaychamber 11 also comprises a pad or matrix 10 of a positively-chargednylon matrix having a pore size of about 0.8 μm. The upper limit on poresize of matrix 10 is chosen to inhibit or prevent blood cell entry intothe matrix. The matrix is positioned in blood glucose assay chamber 11to be in fluidic contact with depression 11A, more preferably coveringdepression 11A, and most preferably having a surface area greater thanthe surface area of depression 11A. The matrix was further impregnatedwith immobilized reagents 11B which produce a detectable productproportional to the amount or concentration of glucose in a bloodsample. Most preferably, the detectable product is a colored product11C, i.e., a product absorbing light at a detectable, most preferably avisible, wavelength.

[0274] Lysis metering chamber 2 is fluidly connected to capillary 7controlled by sacrificial valve 7A. Sacrificial wax valve 7A is furtherfluidly connected with wax recrystallization chamber 7B that is 1 mmdeep and has a volumetric capacity sufficient to sequester melted waxfrom a released wax valve and prevent occlusion of the lumen of thecapillary 7 controlled by the valve. Capillary 7 is 0.25 mm deep, has across-sectional diameter of 0.25 mm, extends 0.1 cm from lysis meteringchamber 2 and is fluidly connected to blood lysis chamber 16. Bloodlysis is contained within the mixing chamber Blood lysis chamber 16 is2.3 mm deep, has a cross-sectional diameter of 1 cm, is positioned 3 cmfrom the center of rotation, and contains 45 μL of blood lysis solution(0.1% Triton-X100 in 50 mM Tris, pH 9.5).

[0275] Blood lysis chamber 16 is fluidly connected at a distal aspect tocapillary 17 controlled by sacrificial valve 18. Capillary 17 is about0.25 mm deep and has a cross-sectional diameter of about 0.25 mmSacrificial wax valve 18 is further fluidly connected with waxrecrystallization chamber 18A that is about 0.5 mm deep and has avolumetric capacity sufficient to sequester melted wax from a releasedwax valve and prevent occlusion of the lumen of the capillary 17controlled by the valve. Capillary 17 is fluidly connected to secondarymetering structure 19. Secondary metering structure 19 is 1.5 mm deep,and is positioned 3.8 cm from the center of rotation. Secondary meteringstructure 19 is constructed to comprise three sections. A first section20 comprises a throwaway section that is used to discard the firstsample and taking the second sample to provide a better sample foranalysis, having a volumetric capacity of about 6.4 μL. Throw awaysection 20 is arranged proximal to the entry position of capillary 17and is separated from a metering section 21 by a septum that extendsfrom the distal wall of the structure to a position just short of theproximal wall of the structure. This arrangement produces a fluidconnection between throwaway section 20 and the metering section 21.Metering section has a volumetric capacity of about 6.4 μL and isfluidly connected to an overflow section 24 having an excess volumetriccapacity of about 90 μL. The volumetric capacity of the overflow sectionis sufficient to accommodate the largest blood fluid volume applied tothe disk.

[0276] Capillary 25 is in fluid connection with secondary meteringstructure 21 at the distal wall of the metering section. Capillary 25 isabout 0.1 5 cm deep and has a cross-sectional diameter of about 0.5 mmand is connected to boronate affinity matrix chamber 28. Capillary 25 isfluidly connected to sacrificial wax valve 26 that is further fluidlyconnected with wax recrystallization chamber 26A Wax recrystallizationchamber 26A is about 0.3 cm deep and has a volumetric capacitysufficient to sequester melted wax from a released wax valve and preventocclusion of the lumen of the capillary 25 controlled by the valve.

[0277] Boronate affinity matrix chamber 28 is about 0.15 cm deep, has across-sectional diameter of about 0.3 cm and is positioned about 4.8 cmfrom the axis of rotation. Boronate affinity matrix chamber 28 is filledwith boronate-functionalized agarose beads (Isolab, $SG-6220) having amean diameter of about 60 μm; the beads are maintained in the chamber 28using a porous frits 29. Boronate affinity matrix chamber 28 is furtherfluidly connected to capillary 31. Capillary 31 is about 0.25 mm deepand has a cross-sectional diameter of about 0.25 mm and is connected tocolumn wash buffer reservoir 30. Column wash buffer reservoir 30 isabout 0.25 cm deep and has a cross-sectional diameter of about 2 cm andis positioned about 3.6 cm from the axis of rotation, more proximal thanboronate affinity matrix chamber 28. Column wash buffer reservoir 30comprises about 290 μL of column wash buffer comprising a solution ofasparagine, magnesium chloride, taurine and D,L-methionine (Isolab,#SG-6220). Fluid flow through capillary 31 is connected to sacrificialvalve 32. Sacrificial wax valve 32 is further fluidly connected with waxrecrystallization chamber 32A that is about 0.5 mm deep and has avolumetric capacity sufficient to sequester melted wax from a releasedwax valve and prevent occlusion of the lumen of the capillary 32controlled by the valve.

[0278] Boronate affinity matrix chamber 28 is further fluidly connectedto capillary 37. Capillary 37 is about 0.5 mm deep and has across-sectional diameter of about 0.5 mm and is connected to samplecollection cuvette array 12. Sample collection cuvette array 12 is about0.25 cm deep and has a cross-sectional diameter of about 2.1 cm and ispositioned about 5 cm from the axis of rotation. Sample collectioncuvette array 12 is separated into a multiplicity of individualchambers, each separated from one another by septa that extend from thedistal wall of the cuvettes to a position adjacent to the proximal wallof the cuvettes, so that a fluid passage 50 is maintained between eachof the cuvettes. The fluid passage 50 is formed by the back (proximalwall) of the sample collection cuvette array 12 and the row of septaseparating each of the sections of the sample collection cuvettes 12.Capillary 33 is fluidly connected to sample collection cuvette array 12at a position adjacent to the proximal wall of the array and directed tothe cuvette most proximal to the boronate affinity matrix chamber 28.

[0279] Capillary 22 is fluidly connected to secondary metering structure19. Capillary 22 is about 0.25 mm deep and has a cross-sectionaldiameter of about 0.25 mm and is connected to secondary meteringstructure 19 at a position between the metering section and the overflowsection. Capillary 22 is further fluidly connected with total hemoglobinread chamber 23. Total hemoglobin read chamber 23 is about 0.25 cm deep,is positioned about 5 cm from the center of rotation, and has avolumetric capacity of about 50 μL. Total hemoglobin read chamber 23 ispositioned radially more distal from the center of rotation thansecondary metering structure 19, and comprises a read window translucentto light having a wavelength of 300-1000 nm. In addition, there is nocapillary or sacrificial valving controlling fluid flow in capillary 23.

[0280] The platform also comprises control sample read cuvettes 13 and14, advantageously positioned in proximity to total hemoglobin readchamber 23. Control sample read cuvettes 13 and 14 are each about 0.25cm deep, positioned about 5 cm from the center of rotation, and have avolumetric capacity of about 50 μL. Control sample read cuvettes 13 and14 comprise a read window translucent to light having a wavelength of410 nm. Control sample read cuvettes 13 and 14 are not fluidly connectedto any other structure on the platform.

[0281] Air displacement channels 33 and capillary junction(s) 34, thatpermit venting of air displaced by fluid movement on the platform, arefluidly connected to the components of the platform to permit unimpededfluid flow.

[0282] As illustrated in FIGS. 12 A through 12Q, in the use of thisplatform a volume of blood about 30 μL is applied to entry port 1. Bloodenters lysis subvolume 2 and lateral passageway 3 under the influence ofgravity and capillary forces in the absence of rotation of the platform,as shown in FIG. 12A. Upon rotation of the platform at a firstrotational speed f₁ of 400-600 rpm, blood completely fills lysissubvolume 2 and also flows through passageway 3 and into blood glucosemetering chamber 4 and overflow chamber 5, shown in FIG. 12B. Blood isretained in blood glucose chamber 4 either due to capillary pressure orby a sacrificial valve 6, most preferably a wax valve. Similarly, bloodis retained in blood lysis subvolume 2 by a valve, most preferably asacrificial valve 7. Excess blood flows at rotational speed f₁ throughoverflow channel 8 and into overflow chamber 9, shown in FIGS. 12C and12D. Typical values for the first rotational speed are an accelerationof about 20 to 60 rpm/sec to a final radial velocity of about 600 rpm.

[0283] After blood is metered and excess blood delivered to overflowchamber 9, the rotational speed of the disc is reduced to a rotationalspeed f_(1a) of 600 to 100 rpm, typically 60 rpm, to perform a blankingmeasurement on sample collection cuvette array 12, total hemoglobin readchamber 23 and blood glucose assay chamber 11. Measurements of blankingcuvettes 13 and 14 are also advantageously performed.

[0284] The disc is then accelerated to a second rotational speed f₂greater than f₁, and typically in the range of 800 rpm to about 1000rpm. At this speed, capillary valve 6 is overcome or sacrificial valve 6is released, and about 16 μL of blood from blood glucose meteringchamber 4 flows through capillary 15 and into blood glucose assaychamber 11, shown in FIG. 12E. Upon entering assay chamber 11, bloodfluid components are forced into absorbent matrix 10 through depression11A. The blood fluid is incubated in matrix 10 for a time sufficient forthe reagents 10A to produce a colored product 10B in an amountproportional to the amount of glucose in the blood fluid sample. Thedisc is slowed, typically to a rotational speed f₄ of about 100 rpm, forglucose data acquisition using reflectance spectrometry; dataacquisition as the disc is spinning down also enables to instrument toset t=0 for the assay, based on a decrease in reflectance when thematrix 10 is wet by the blood fluid components and hence the matrix'sscattering decreases. Development of colored product 10B is shown inFIG. 12G.

[0285] The disc is then accelerated to rotational speed f₃ of about 1000rpm, with release of sacrificial valve 7A and fluid flow of about 5-6.4μL of blood from metered subvolume 2 through capillary 7 and into bloodlysis chamber 16 containing about 40 μL of blood lysis buffer. This isshown in FIG. 12H. The mixture of blood and blood lysis buffer in bloodlysis chamber 16 is mixed by agitation, wherein the platform isaccelerated repeatedly +250 rpm/sec to −250 rpm/sec (wherein “+” and “−”indicate acceleration in different directions), typically 250-500rpm/sec, over a time period of about 2 min, typically 1-3 min, as shownin FIGS. 12I and 12J.

[0286] The disc then is accelerated to a rotational speed f₅ of 1000 rpmand sacrificial valve 18 is released. Lysed blood from blood lysischamber 16 flows through capillary 17 and into secondary meteringstructure 19, as shown in FIG. 12K. The lysed blood solutionsequentially fills throwaway section 20, which is used as a trap forcell debris, metering section 21 and excess lysed blood then fillsoverflow section 24. Filling of metering chamber 21 is immediatelyfollowed by fluid flow through capillary 22 and filling of totalhemoglobin read chamber 23. The disc is spun at rotational speed f₅ fora time sufficient to substantially completely drain blood lysis chamber16. The configuration of the blood fluids on the disc after this spin isshown in FIG. 12L.

[0287] The disc is then decelerated to a rotational speed of 750 rpm,and sacrificial valve 26 is released. A metered volume of about 6.4 μLof lysed blood from metering section 21 flows through capillary 27 andinto boronate affinity matrix chamber 28 (shown in FIG. 12M). The lysedblood solution is allowed to incubate in the chamber for a time of about1 min, sufficient for glycated hemoglobin to bind to the matrix. Thisaspect of the disc is illustrated in FIG. 12N.

[0288] The disc is then accelerated to a rotational speed of 1000 rpm,and sacrificial valve 32 is released. A volume of about 290 μL of columnwash buffer (Isolab #SG-6220) flows from wash buffer reservoir 30 thoughcapillary 31 and into boronate affinity matrix chamber 28 (shown in FIG.12O). The wash buffer displaces the non-glycated hemoglobin and othercomponents of the lysed blood fluid from the affinity column matrix andinto sample collection cuvette array 12. FIGS. 12P through 12Q showsequential filling of the individual cuvettes in sample collectioncuvette array 12. The rotation speed of the disc is reduced, to 60 rpmfor sample collection cuvette array 12 and total hemoglobin read chamber23 to be interrogated spectrophotometrically. The glycated fraction ofthe blood sample is determine algorithmically by subtracting thenon-glycated hemoglobin fraction in sample collection cuvette array 12from the total hemoglobin detected in total hemoglobin read chamber 23.

EXAMPLE 4 Blood Glucose Assay—Chemistries

[0289] Microsystems platforms as provided by the invention were used toperform blood glucose and glycated hemoglobin assays as describedherein.

Blood Glucose Assay

[0290] A series of blood glucose assays were performed to determine theprecision of repeated glucose assays using the Microsystems platforms ofthe invention.

[0291] The assays were performed on a Microsystems platform according toExamine 3 using a round reagent cuvette (113). 60-70 μL of human wholeblood was applied to the disk, and 15 μL metered into the meteringcapillary. A glucose reagent pad obtained from LifeScan was used forperforming the glucose determination. Optical absorbance readings wereobtained at 430 nm, 590 nm and 660 nm on the unreacted reagent pad as acontrol. Blood was released into the reaction chamber and optical datagathered at one-second intervals for about 1 min. Absorbance at 590 nmwas specific for the amount of glucose in the sample; absorbance at 430nm was specific for hemoglobin in the sample; and absorbance at 660 nmwas used to detect non-specific background absorbance, as describedabove.

[0292] A total of seven assays were performed for each of two samplesand the amount of glucose (in mg/dL) determined as follows: Sample 1Sample 2 Glucose mg/dL 136.0 113.2 % CV 4.4% 4.5%

[0293] These assays were repeated as above using a Microsystems platformaccording to Example 1 comprising a rectangular reagent cuvette (113). ABiodyne-B membrane (Pall) was impregnated with glucose oxidase reagentsas described above and used for performing the glucose determination. Inthese embodiments, chamber 113 had dimensions of 4 mm×5 mm.

[0294] The glucose determination was performed exactly as described inthe first series of experiments. Standard blood glucose assays were runon these samples in parallel to compare the results obtained with theMicrosystems platforms of the invention with conventional assays. Theseresults were as follows: mg/dL Reference Method mg/dL Invention 46.861.9 85.9 76.8 107.0 115.8 149.0 144.2

[0295] These results established that the microsystems platforms of theinvention were capable of determining blood glucose concentrations.

Correction for Interfering Substances

[0296] In addition, assays were performed on a Microsystems platform toExample 3 using a round reagent cuvette (113) and the glucose resultsobtained were corrected for hemoglobin interferences as discussedherein. In these experiments, the spectrophotometric results obtainedwere used to calculate blood glucose with and without correction for thehemoglobin absorbance signal at 590 nm. The correction factor wasdetermined empirically using samples comprised of phosphate bufferedsaline to which known qualities of glucose and/or hemoglobin were added.The resulting samples were assayed on the Microsystems platformaccording to Example 3 using a round reagent cuvette (113) to give thefollowing data: Glucose Hemoglobin Concentration ConcentrationReflectance Reflectance Sample (mg/dL) (mg/dL) (590 nm) (430 nm) A 0 00.0152 −0.0204 B 0 1.25 0.2116 0.3041 C 75 5 1.2767 0.3452 D 78 0 0.95760.1311

[0297] For glucose chromagen alone, Sample D, the Reflectance(430 nm) is16.1% of the Reflectance(590 nm) [(0.131-(-0.0204)/(0.9576-0.152)]. Forhemoglobin alone, Sample B, the Reflectance(590 nm) is 60.5% of theReflectance (430) [(0.2116-0.0152)/(0.3041-(-0.0204))]. For thecombination of glucose and hemoglobin, Sample C, the Reflectance(430 nm)is the sum of the Reflectance (430 nm) due to the hemoglobin plus 16.1%of the Reflectance(590 nm) due to the glucose chromagen; similarly, theReflectance(590 nm) is the sum of the Reflectance(590 nm) due to theglucose chromagen plus 60.5% of the Reflectance(430 nm) due to thehemoglobin. Using standard algebraic methods, the factor to be used wascalculated to be:

[0298] Reflectance(590 nm) due to glucose chromagen=Reflectance(590nm)−F×Reflectance(430 nm) where F=0.274

[0299] The results shown in FIGS. 14A and 14B illustrate the differencein the accuracy of the data obtained with and without correcting for thehemoglobin absorbance signal.

Glycated Hemoglobin Assay

[0300] A series of glycated hemoglobin assays were performed todetermine the precision of repeated binding of glycated hemoglobin bymeasuring the amount of hemoglobin put on a phenyl boronate column andthe amount of (non-glycated) hemoglobin that could be eluted from thecolumn.

[0301] These experiments were performed using a microsystems platform ofthe invention as disclosed in Example 3; the phenyl boronate columnmaterial was from a kit obtained from IsoLab Glyc-Affin GHb. 60-70 μL ofhuman whole blood was applied to the disk, and 5 μL metered into andmixed with 45 μL of lysis buffer in the mixing chamber of the platform.5 μL of the lysed blood was metered onto the phenyl boronate column andnon-glycated hemoglobin eluted with 290 μL of elution buffer. Thereflective absorbance of the lysed blood and the eluted non-glycatedhemoglobin was determined spectrophotometrically at 430 nm and used tocalculate the amount of non-glycated hemoglobin in the sample.

[0302] Seven separate assays were performed on each of 2 differencewhole blood samples. These results are as follows: Sample 1 Sample 2 %NGHb, reference method 93 90 % NGHb, invention 93 92 % CV 1.2% 1.7%

DNA Binding Assays

[0303] These experiments were performed to show that the microsystemsplatforms of the invention could efficiently isolate plasmid DNA from asample. In these experiments, diatomeceous earth was used tospecifically bind plasmid DNA, and the results were compared with astandard plasmid DNA extraction kit (Qiagen).

[0304] In these experiments, the Qiagen assay was performed until thebacterial culture had been disrupted with chaotropic salts. At thispoint, the solution was split in half, and one half assayed using theQiagen reagents and columns, and the other half 300 μL assayed using amicrosystem platform adapted from the one described in Example 2. Inthis embodiment, the diatomeceous earth column was loaded onto theplatform and the sample applied thereto by centrifugation at about 800rpm. The column was washed twice with 100 μL of Qiagen buffer PE atabout 850 rpm, and the plasmid DNA was then eluted twice, with 50 μL ofQiagen Buffer EB, once at 600 rpm and again at at 900 rpm. The elutedplasmid DNA from each preparation was precipitated by the addition ofice cold ethanol to 70% and the DNA pellet collected by centrifugationand resuspended in 50 μL of 10 mM Tris-HCl, pH 8. 15 μL were analyzed byconventional agarose gel electrophoresis, stained with ethidium bromideand osverved under ultraviolet light illumination.

[0305] The results of these assays are shown in FIG. 15, and illustratethe use of the Microsystems platforms of the invention for isolatingplasmid DNA from a biological fluid sample.

[0306] It should be understood that the foregoing disclosure emphasizescertain specific embodiments of the invention and that all modificationsor alternatives equivalent thereto are within the spirit and scope ofthe invention as set forth in the appended claims.

We claim:
 1. A microsystem platform for separating an analyte from afluid sample, comprising a) a rotatable platform, comprising a substratehaving a first flat, planar surface and a second flat, planar surfaceopposite thereto, each surface comprising a center about which theplatform is rotated, wherein the first surface comprises in combinationb) an entry port comprising a depression in the first surface having avolumetric capacity of about 1 to about 150 μL, that is fluidlyconnected by a first microchannel with c) a mixing chamber positioned onthe platform farther from the center of rotation than the entry port;the platform further comprising i) a reagent reservoir comprising liquidreagents for preparing the fluid sample for the analyte separationassay, wherein the reagent reservoir if fluidly connected to the mixingchamber by a second microchannel, the platform further comprising d) asecondary metering chamber comprising a first metering portion and asecond metering portion each defining a volume of the fluid andseparated by a septum that extends from a position in the chamberfarthest from the center of rotation to a position just short of achamber wall closest to the center of rotation, wherein the end of theseptum and the chamber wall define a fluid connection between the firstand second metering portions, the metering chamber further comprising anoverflow portion that is separated from the second metering portion by aseptum that extends from a position in the chamber farthest from thecenter of rotation to a position just short of a chamber wall closest tothe center of rotation, and wherein the first portion of the meteringchamber is fluidly connected by a third microchannel to e) an analyteseparation assay chamber further comprising i) an analyte bindingmatrix, wherein the analyte specifically binds to the matrix and isretained in the separation chamber thereby, wherein the analyteseparation assay chamber is further fluidly connected by a fourthmicrochannel with a separation matrix preparation buffer reservoircontaining a preparation buffer and the analyte separation assay chamberis further fluidly connected by a fifth microchannel with a separationmatrix wash buffer reservoir containing a separation matrix wash buffer,wherein each of the preparation buffer and wash buffer reservoirs arepositioned closer to the center of rotation than the analyte separationassay chamber; and wherein the second metering portion of the secondarymetering chamber is fluidly connected by a sixth microchannel with f) aread window that is further fluidly connected to the analyte separationassay chamber by a seventh microchannel and is further fluidly connectedby a eighth microchannel to g) a waste reservoir wherein rotation of theplatform motivates the fluid sample from the entry port into the mixingchamber and the reagents from the reagent reservoir into the mixingchamber to provide a sample reagent mixture, and wherein rotation of theplatform motivates the sample reagent mixture from the mixing chamberinto the secondary metering chamber, wherein the first metering portionis filled before the second metering portion and the second meteringportion is filled before the overflow portion; and wherein rotation ofthe platform motivates separation matrix preparation buffer through theanalyte separation assay chamber, through the read chamber and into thewaste reservoir, and wherein rotation of the platform motivates thevolume of the sample reagent mixture from the first metering portion ofthe secondary metering chamber through the analyte separation assaychamber whereby analyte in the sample reagent mixture binds to theanalyte binding matrix; and wherein rotation of the platform motivatesanalyte separation matrix wash buffer through the analyte separationassay chamber, thereby displacing the sample reagent mixture through theread window and into the waste reservoir; and wherein rotation of theplatform motivates the volume of the sample reagent mixture in thesecond metering portion of the secondary metering chamber into the readwindow and displaces the eluate from the analyte separation assaychamber from the read window.
 2. The microsystem platform of claim 1wherein the analyte binding matrix is an inositol phosphate-derivedmembrane.
 3. The microsystem platform of claim 1 further comprising e) ametering capillary and an overflow capillary, each being fluidlyconnected with the entry port, wherein each capillary defines across-sectional area of about 0.02 mm to about 1 mm in diameter, andwherein each capillary extends radially from the center of the platformand defines a first end proximally arrayed towards the center of theplatform and a second end distally arrayed from the center of theplatform, wherein the proximal end of each capillary defines a curvedopening; wherein the metering capillary defines a volume of the fluidand wherein the metering capillary is fluidly connected with the mixingchamber and wherein the overflow capillary is fluidly connected with f)an overflow chamber having a depth equal to or greater than the overflowcapillary and positioned radially more distant from the center of theplatform than the mixing chamber and the entry port, wherein a capillaryjunction is formed at the junction of the metering capillary and themixing chamber and at the junction of the overflow capillary and theoverflow chamber, whereby fluid placed in the entry port flows bycapillary action to the junction of the metering capillary and themixing chamber, and excess fluid flows by capillary action to thejunction of the overflow capillary and the overflow chamber; and whereinrotation of the platform at a first rotation speed motivates fluiddisplacement in the overflow capillary into the overflow chamber but notfluid displacement in the metering capillary, whereby rotation of theplatform at the first rotational speed drains the fluid from the entryport into the overflow chamber; and wherein rotation of the platform ata second rotation speed that is greater than the first rotational speedmotivates fluid displacement of the volume of the fluid in the meteringcapillary into the mixing chamber; and wherein each of the assay chamberand overflow chamber also comprise air displacement channels whereby airdisplaced by fluid movement is vented to the surface of the platform. 4.The microsystem platform of claim 1, further comprising j) a sacrificialvalve in the fourth, fifth, sixth, or seventh microchannels, whereinrelease of the sacrificial valve permits fluid flow through themicrochannel when the platform is rotated at a non-zero rotationalspeed.
 5. The microsystem platform of claim 4 wherein the sacrificialvalve is a solid, semi-solid or viscous liquid hydrocarbon, or aplastic.
 6. The microsystem platform of claim 5 further comprising aheating element in the platform in thermal contact with the sacrificialvalve, wherein heating the heating element releases the sacrificialvalve.
 7. The microsystem platform of claim 1, wherein the read chambercomprises a top surface that is translucent.
 8. The microsystem platformof claim 1 wherein the fluid sample is blood.
 9. A microsystem platformfor separating an analyte from a fluid sample, comprising a) a rotatableplatform, comprising a substrate having a first flat, planar surface anda second flat, planar surface opposite thereto, each surface comprisinga center about which the platform is rotated, wherein the first surfacecomprises in combination b) an entry port comprising a depression in thefirst surface having a volumetric capacity of about 1 to about 150 μL,that is fluidly connected by a first microchannel with c) a mixingchamber positioned on the platform farther from the center of rotationthan the entry port and comprising liquid reagents for preparing thefluid sample for the analyte separation assay, wherein the mixingchamber is fluidly connected by a second microchannel with d) asecondary metering chamber comprising a first metering portion and asecond overflow portion wherein the first metering portion defines avolume of the sample reagent mixture, wherein the first metering portionand the second overflow portion are separated by a septum that extendsfrom a position in the chamber farthest from the center of rotation to aposition just short of a chamber wall closest to the center of rotation,wherein the end of the septum and the chamber wall define a fluidconnection between the first metering portion and the overflow portion,and wherein the fluid connection between the first metering portion andthe overflow portion is fluidly connected by a third microchannel to aread chamber positioned radially more distant from the center ofrotation than the secondary metering chamber, and wherein the firstmetering portion of the secondary metering chamber is fluidly connectedby a fourth microchannel to e) an analyte separation assay chamberfurther comprising i) an analyte binding matrix, wherein the analytespecifically binds to the matrix and is retained in the separationchamber thereby, wherein the analyte separation assay chamber is furtherfluidly connected by a fifth microchannel with a separation matrixbuffer reservoir containing a buffer, wherein the matrix bufferreservoir is positioned closer to the center of rotation than theanalyte separation assay chamber; and wherein the analyte separationassay chamber is further fluidly connected by a sixth microchannel withf) a read window manifold comprising a series of chambers separated bysepta and arranged linearly and adjacently on the surface of theplatform away from the position of the fluid connection of the manifoldwith the fifth microchannel; wherein rotation of the platform motivatesthe fluid sample from the entry port into the mixing chamber to a samplereagent mixture, and wherein rotation of the platform motivates thesample reagent mixture from the mixing chamber into the secondarymetering chamber, wherein the first metering portion is filled beforethe overflow portion and the read chamber is filled by fluid flowthrough the second microchannel before the overflow portion is filled;and wherein rotation of the platform motivates separation matrixpreparation buffer through the analyte separation assay chamber and intothe read chamber manifold, and wherein rotation of the platformmotivates the volume of the sample reagent mixture from the firstmetering portion of the secondary metering chamber through the analyteseparation assay chamber whereby analyte in the sample reagent mixturebinds to the analyte binding matrix; and wherein rotation of theplatform motivates analyte separation matrix wash buffer through theanalyte separation assay chamber, thereby displacing the sample reagentmixture into the read manifold.
 10. The microsystem platform of claim 9further comprising g) metering capillary and an overflow capillary, eachbeing fluidly connected with the entry port, wherein each capillarydefines a cross-sectional area of about 0.02 mm to about 1 mm indiameter, and wherein each capillary extends radially from the center ofthe platform and defines a first end proximally arrayed towards thecenter of the platform and a second end distally arrayed from the centerof the platform, wherein the proximal end of each capillary defines acurved opening; wherein the metering capillary defines a volume of thefluid and wherein the metering capillary is fluidly connected with themixing chamber and wherein the overflow capillary is fluidly connectedwith h) overflow chamber having a depth in the platform equal to orgreater than the overflow capillary and positioned radially more distantfrom the center of the platform than the mixing chamber and the entryport, wherein a capillary junction is formed at the junction of themetering capillary and the mixing chamber and at the junction of theoverflow capillary and the overflow chamber, whereby fluid placed in theentry port flows by capillary action to the junction of the meteringcapillary and the mixing chamber, and excess fluid flows by capillaryaction to the junction of the overflow capillary and the overflowchamber; and wherein rotation of the platform at a first rotation speedmotivates fluid displacement in the overflow capillary into the overflowchamber but not fluid displacement in the metering capillary, wherebyrotation of the platform at the first rotational speed drains the fluidfrom the entry port into the overflow chamber; and wherein rotation ofthe platform at a second rotation speed that is greater than the firstrotational speed motivates fluid displacement of the volume of the fluidin the metering capillary into the mixing chamber; and wherein each ofthe assay chamber and overflow chamber also comprise air displacementchannels whereby air displaced by fluid movement is vented to thesurface of the platform.
 11. The microsystem platform of claim 9,further comprising j) a sacrificial valve in the third, fourth, fifth,or sixth microchannels, wherein release of the sacrificial valve permitsfluid flow through the microchannel when the platform is rotated at anon-zero rotational speed.
 12. The microsystem platform of claim 11wherein the sacrificial valve is a solid, semi-solid or viscous liquidhydrocarbon, or a plastic.
 13. The microsystem platform of claim 12further comprising a heating element in the platform in thermal contactwith the sacrificial valve, wherein heating the heating element releasesthe sacrificial valve.
 14. The microsystem platform of claim 9, whereinthe read chamber comprises a top surface that is translucent.
 15. Themicrosystem platform of claim 9 wherein the fluid sample is blood.
 16. Amicrosystem platform for separating an analyte from a fluid sample,comprising a) a rotatable platform, comprising a substrate having afirst flat, planar surface and a second flat, planar surface oppositethereto, each surface comprising a center about which the platform isrotated, wherein the first surface comprises in combination b) an entryport comprising a depression in the first surface having a volumetriccapacity of about 1 to about 150 μL, that is fluidly connected by afirst microchannel with c) an assay chamber fluidly connected with theentry port, the reaction chamber further comprising i) a porous matrixcomprising reagents for performing an analyte detection assay wherein afluid sample applied to the entry port is delivered to the assay chamberthrough the capillary microchannel by rotation of the platform, andwherein delivery of the fluid sample to the assay chamber initiates theanalyte detection assay; wherein the entry port is further fluidlyconnected with d) a mixing chamber positioned on the platform fartherfrom the center of rotation than the entry port and comprising liquidreagents for preparing the fluid sample for the analyte separationassay, wherein the mixing chamber is fluidly connected by a secondmicrochannel with e) a secondary metering chamber comprising a firstmetering portion and a second overflow portion wherein the firstmetering portion defines a volume of the sample reagent mixture, whereinthe first metering portion and the second overflow portion are separatedby a septum that extends from a position in the chamber farthest fromthe center of rotation to a position just short of a chamber wallclosest to the center of rotation, wherein the end of the septum and thechamber wall define a fluid connection between the first meteringportion and the overflow portion, and wherein the fluid connectionbetween the first metering portion and the overflow portion is fluidlyconnected by a third microchannel to a read chamber positioned radiallymore distant from the center of rotation than the secondary meteringchamber, and wherein the first metering portion of the secondarymetering chamber is fluidly connected by a fourth microchannel to f) ananalyte separation assay chamber further comprising i) an analytebinding matrix, wherein the analyte specifically binds to the matrix andis retained in the separation chamber thereby, wherein the analyteseparation assay chamber is further fluidly connected by a fifthmicrochannel with a separation matrix buffer reservoir containing abuffer, wherein the matrix buffer reservoir is positioned closer to thecenter of rotation than the analyte separation assay chamber; andwherein the analyte separation assay chamber is further fluidlyconnected by a sixth microchannel with g) a read window manifoldcomprising a series of chambers separated by septa and arranged linearlyand adjacently on the platform away from the position of the fluidconnection of the manifold with the fifth microchannel; wherein rotationof the platform motivates the fluid sample from the entry port into themixing chamber to a sample reagent mixture, and wherein rotation of theplatform motivates the sample reagent mixture from the mixing chamberinto the secondary metering chamber, wherein the first metering portionis filled before the overflow portion and the read chamber is filled byfluid flow through the second microchannel before the overflow portionis filled; and wherein rotation of the platform motivates separationmatrix preparation buffer through the analyte separation assay chamberand into the read chamber manifold, and wherein rotation of the platformmotivates the volume of the sample reagent mixture from the firstmetering portion of the secondary metering chamber through the analyteseparation assay chamber whereby analyte in the sample reagent mixturebinds to the analyte binding matrix; and wherein rotation of theplatform motivates analyte separation matrix wash buffer through theanalyte separation assay chamber, thereby displacing the sample reagentmixture into the read manifold.
 17. The microsystem platform of claim 16wherein the analyte binding matrix is an inositol phosphate-derivedmembrane.
 18. The microsystem platform of claim 16, further comprisingh) a sacrificial valve in the fourth, fifth, sixth, or seventhmicrochannels, wherein release of the sacrificial valve permits fluidflow through the microchannel when the platform is rotated at a non-zerorotational speed.
 19. The microsystem platform of claim 18 wherein thesacrificial valve is a solid, semi-solid or viscous liquid hydrocarbon,or a plastic.
 20. The microsystem platform of claim 19 furthercomprising a heating element in the platform in thermal contact with thesacrificial valve, wherein heating the heating element releases thesacrificial valve.
 21. The microsystem platform of claim 16, wherein theread chamber comprises a top surface that is translucent.
 22. Themicrosystem platform of claim 16 wherein the fluid sample is blood.